INTER- AND INTRA- VARIATION IN SECONDARY METABOLITES

FROM OCTOCORALS OF THE GENUS

PSEUDOPTEROGORGIA: ANTIOXIDANT ACTIVITY AND A POTENTIAL

ECOLOGICAL ROLE

by

MAIA STAPLETON MUKHERJEE

(Under the direction of William K. Fitt)

ABSTRACT

Trends of the past century suggest that bleaching events may become more frequent and severe as the climate continues to warm, exposing coral reefs to an increasingly hostile environment. The combination of higher than average temperatures

(as small as 1°C–2°C) and intense light can lead to mass events, which result in apoptosis and/or the expulsion of the zooxanthellae from the host tissue. The decline of coral reefs is significant, because coral reefs maintain high levels of

biodiversity, provide habitats for coastal fisheries, protect shorelines from erosion, and

may contain potential pharmacological compounds.

A striking difference between Caribbean octocorals and other zooxanthellate

cnidarians is their seemingly greater resistance of bleaching. While there have been

numerous reports of scleractinian coral bleaching throughout the world’s tropical oceans,

there have been far fewer observations of bleaching among octocorals. If the increasing incidence of bleaching events continues, the relative insensitivity of most gorgonians to high temperatures may have important consequences for Caribbean reefs.

This research attempts to examine whether secondary metabolites from the genus

Pseudopterogorgia act as antioxidants and thus mediate thermal and UV induced oxidative stress and thereby protect the coral-algal symbiosis. Chemical diversity in soft may have an adaptive value in mediating ecological interactions in environments. Extracts from Pseudopterogorgia sps have differing antioxidant potentials as seen using the FRAP assay. Diterpenes isolated from P elisabethae exhibit antioxidant potential in FRAP and pseudopterosins scavenge superoxide radicals generated in mouse

neuronal cells deprived of NGF and prevent apoptosis. Most significantly, pseudopterosins (and especially the aglycone) reduce the loss of quantum photosynthetic

yield in zooxanthellae exposed to excess heat and light, and reduced significantly the

oxidation of the probe DCFH in the zooxanthellae/ micro-plate based assay.

Index Words: Coral bleaching, Photoinhibition, Octocoral, Zooxanthellae, Antioxidant, Pseudopterogorgia, Pseudopterosin, Natural product, FRAP, DCFH-DA, Nerve Growth Factor, Diterpene

INTER- AND INTRA- SPECIES VARIATION IN SECONDARY METABOLITES

FROM CARIBBEAN OCTOCORALS OF THE GENUS

PSEUDOPTEROGORGIA: ANTIOXIDANT ACTIVITY AND A POTENTIAL

ECOLOGICAL ROLE

by

MAIA STAPLETON MUKHERJEE

B.A., Michigan State University, 1987

B.A., Michigan State University, 1988

M.A., New York University, 1998

M.S., Florida Atlantic University, 2005

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in

Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2008

©2008

Maia Stapleton Mukherjee

All Rights Reserved

INTER- AND INTRA- SPECIES VARIATION IN SECONDARY METABOLITES

FROM CARIBBEAN OCTOCORALS OF THE GENUS

PSEUDOPTEROGORGIA: ANTIOXIDANT ACTIVITY AND A POTENTIAL

ECOLOGICAL ROLE

by

MAIA STAPLETON MUKHERJEE

Major Professor: William K. Fitt

Committee: James W. Porter James L. Franklin James L. Shelton C. Ronald Carroll

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia December, 2008

DEDICATION

For my father and mother; we were but stones; your light made us stars.

For Kevin, who reminds me every day that the world is full of wondrous discoveries yet to be made.

iv ACKNOWLEDGEMENTS

Thanks to my advisor, Dr. William Fitt, and the members of my committee, Dr. Jim

Porter, Dr. Jim Franklin, Dr. Jay Shelton and Dr. Ron Carroll for all the support and advice.

Thanks to Dr. Gregory Schmidt and Dr. Bill Fitt for allowing me to use cultures of

Symbiodinium spp and the PAM Fluorometer. Thanks to Clint Oakley for assisting me with the zooxanthellae based assays.

Thanks to Rebecca Kirkland and Jim Franklin for performing the assays with mouse sympathetic neurons.

Thanks to Dr. Prasoon Gupta and Dr. Lyndon West for the structural elucidation and characterization of pseudopterosins.

Additional thanks to Dr. West his support and for generously allowing me to use the facilities in his lab to conduct my research.

Thanks to Dr. Howard Lasker for supporting my field work, for generously sharing his expertise in octocoral biology and ecology, and all his advice and encouragement.

Finally a very special thanks to the Mukherjees; mom, dad, Joia, Janam, Che, and Kevin-

“If not for you, winter would have no spring, couldn't hear the robin sing, I just wouldn't have a clue, anyway it wouldn't ring true, if not for you.”

v TABLE OF CONTENTS

ACKNOWLEDGEMENTS……………………………………………………… v

LIST OF TABLES………………………………………………………………... viii

LIST OF FIGURES………………………………………………………………. x

CHAPTER

1 Introduction and Background……………………………………….. 1

1.1-Coral reefs and coral bleaching………………………………….. 1

1.2-Natural products………………………………………………….. 10

1.3-Marine natural products……………………...... 13

1.4 Reactive oxygen and antioxidants……………………………….. 26

1.5 The antioxidant potential of extracts and compounds from Pseudopterogorgia sps………………………………………………… 32

2 Intra-specific chemical variability of Pseudopterogorgia elisabethae 37

2.1 Caribbean octocorals……………………………………………... 37

2.2 Terpene Biosynthesis……………………………………………... 38

2.3 Terpenes from Pseudopterogorgia spp…………………………... 40

2.4 Pseudopterosins…………………………………………………… 42

2.5 Variability of pseudopterosin content across a geographic gradient...... 52

vi 3 Antioxidant potential of extracts and pure compounds from Pseudopterogorgia sps………………………………………………………. 68

3.1 Antioxidants and marine organisms…………………………….. 68

3.2 Ferric reducing antioxidant power (FRAP) assay……………… 70

4 In vivo antioxidant activity of extracts and pure compounds from Pseudopterogorgia…………………………………………………………... 90

4.1 Photosynthesis and oxidative stress……………………………… 90

4.2 Detection of intracellular ROS using 2’,7’- dichlorodihydrofluorescein diacetate………………………………... 92

4.3 Detection of photoinhibition using PAM fluorometry………….. 100

5 A potential pharmacological role for the antioxidants from Pseudopterogorgia sps………………………………………………………. 110

5.1 Oxidative stress and human diseases………………...... 110

5.2 Oxidative stress and neurodengerative diseases………………... 112

5.3 Pseudopterosins promote survival in NGF deprived mouse sympathetic neurons………………………………………………….. 116

5.4 Pseudopterosins suppress ROS formation in NGF deprived sympathetic neurons………………………………………………….. 118

6 Methods and materials………………………………………………... 123

REFERENCES……………………………………………………………………. 133

APPENDICES…………………………………………………………………….. 163

vii LIST OF TABLES

Table 1. Comparison of pseudopterosin A-D and kallolide A Acetate content from P. elisabethae and P. bipinnata expressed as % organic extract... 49

Table 2. Comparison specific activities of pseudopterosin and Kallolide A acetate from larvae treated with antibiotics and antimycotics and those untreated. Average of n = 3 experiments ± st. dev…………………… 51

Table 3. Distribution of pseudopterosins. Sites are grouped by chemotype. Values reported as %w/w of the 75% fraction of the crude extract…………………………………………………………………. 63

Table 4. FRAP assay of the intermediate polarity (75%) fraction of seven species of Pseudopterogorgia. Values given are µM of Trolox / µg extract, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……………….. 76

Table 5. FRAP assay of pure pseudopterosins and related molecules isolated from Pseudopterogorgia elisabethae Values given are µM of Trolox / µg extract, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……………….. 82

Table 6. DCFH-DA assay of the intermediate polarity (75%) fraction of seven species of Pseudopterogorgia. Values given are % reduction in DCF fluorescence versus MeOH control, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD………………………………………………………….. 96

Table 7. DCFH-DA assay of pure pseudopterosins and related molecules isolated from Pseudopterogorgia elisabethae, given as percent fluorescence inhibition versus control cells (MeOH only) n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD………………………...... 98

Table 8. Pairwise comparison of treatments; Tukey’s HSD, p<0.05…………... 106

Table A1. GPS coordinates of Pseudopterogorgia sps. collection sites…………. 163

Table A2. One-way analysis of variance for FRAP assay of 75% fractions from seven species of the genus Pseudopterogorgia shows that means are significantly different amongst the treatments. (Graphpad Prism)……. 163

viii Table A3. One-way analysis of variance for FRAP assay of 15 pseudopterosins isolated from Pseudopterogorgia elisabethae shows that means are significantly different amongst the treatments. The water soluble vitamin E analog Trolox serves as the positive control. (Graphpad Prism)………………………………………………………………….. 164

Table A4. One-way analysis of variance for DCFH-DA - zooxanthellae assay of 75% fractions from seven species of the genus Pseudopterogorgia shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)……………………………………………………… 164

Table A5. One-way analysis of variance for DCFH-DA - zooxanthellae assay of 15 pseudopterosins isolated from Pseudopterogorgia elisabethae shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)……………………………………………………… 165

Table A6. Repeated measures analysis of variance for photoinhibition assay shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)……………………………………………………… 165

Table A7. One-way analysis of variance for survival of mouse sympathetic neurons deprived of NGF and treated with three concentrations (10, 20 and 40 µM) of pseudopterosin K, ps K 2’ac and ps K 4’ ac shows that means are significantly different amongst the treatments………... 166

Table A8. One-way analysis of variance of ROS quantification using MitoSox Red dye………………………………………………………………... 166

Table A9. NMR data for pseudopterosins K 2’ acetate and 4’ acetate, pseudopterosins iso- A, C and D. Recorded in CDCl3 at 500 MHz (TMS as internal standard), chemical shifts, multiplicity and coupling constants (J, Hz) were assigned by means of 1H, 13C NMR and 2D NMR data……………………………………………………………… 167

ix LIST OF FIGURES

Figure 1. Structure of salicylic acid (1) and acetylsalicylic acid (2)…………….. 12

Figure 2. Structures of paclitaxel (3) and doxorubicin (4)………………………. 13

Figure 3. Structures of ara-A (5), ara-C (6) and Prialt® (7)……………………... 15

Figure 4. Structures of bryostatin 1 (8) and ecteinascidin-743 (9)……………… 16

Figure 5. Structures of bipinnatans A-D (10-13)………………………………... 23

Figure 6. Structures of kallolide A (14) and kallolide A acetate (15)…………... 23

Figure 7. Structures of pseudopterosins A-D (17-20)…………………………… 24

Figure 8. Biosynthesis of geranyl, farnesyl, and geranylgeranyl diphosphate (23-25) from isopentenyl diphosphate (21) and dimethylallyl diphosphate(22)……………………………………………………….. 39

Figure 9. Pseudopterosin biosynthesis. Ferns et al., 2005………………………. 43

Figure 10. An example of an agarose gel showing amplification products from larvae, isolated zooxanthellae, and holobiont DNA using zooxanthellae and Pseudopterogorgia specific primers………………. 50

Figure 11. Collection sites of Pseudopterogorgia elisabethae in the Bahamas and Florida. Detail shows the sites clustered along the Little Bahamas Bank…………………………………………………………………… 54

Figure 12. HPLC chromatograms of chemotypes of P. elisabethae-BSS chemotype: A = San Salvador, B = Burrows; BHY chemotype C= Burrows; PKL chemotype, L= Pickles Reef…………………………... 57

Figure 13. HPLC chromatograms of chemotypes of P. elisabethae- D= Cat Island, E= Hog , F= Little San Salvador, G= Bimini……………... 58

x Figure 14. HPLC chromatograms of chemotypes of P. elisabethae- H= Gorda Rock, I= Long Rock, J = AB102, K= Sandy Point…………………… 59

Figure 15. Map of the hydrography of the collections region…………………….. 61

Figure 16. Structures of pseudopterosins G-K, iso A-D and ps G-J aglycone……. 62

Figure 17. An example of a standard curve generated using concentrations of FeSO4……………………………………………………………………………………………………. 73

Figure 18. FRAP analysis of polar (40%), medium-polar (75%) and non-polar (100%) fractions of the crude extract of seven species of the genus Pseudopterogorgia……………………………………………………. 73

Figure 19. Example of a standard curve generate using concentrations of Trolox from 5-100 µM in a FRAP assay……………………………………… 75

Figure 20. FRAP assay of 75% fractions of seven species of Pseudopterogorgia. Values represent µM Trolox/µg extract. Mean of n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD………………………………………………….. 77

Figure 21. Comparison of chromatograms and graphical representations of FRAP analysis of 96-well plate collections of HPLC separation of 75% fraction…………………………………………………………… 79

Figure 22. FRAP assay of 96-well plate collection from HPLC of 75% fraction. For each of four of the chemotypes one chromatogram, corresponding FRAP plate and graph of FRAP values for each well. FRAP values expressed in µM equivalent FeSO4………………………………………………………. 80

Figure 23. FRAP assay pseudopterosins and related molecules from Pseudopterogorgia elisabethae throughout the study region. Values represent µM Trolox/µM pseudopterosin. Mean of n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD………………………………………………….. 83

Figure 24. Structure of Trolox, a synthetic vitamin E analog with potent antioxidant activity……………………………………………………. 84

Figure 25. Structure of carnosic acid and carnosol, phenolic diterpenes from terrestrial plants with potent antioxidant activity……………………... 85

xi Figure 26. Results of the zooxanthellae-based DCFH-DA assay given as percent fluorescence inhibition versus control cells (MeOH only). n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……………………………………….. 97

Figure 27. Results of the zooxanthellae-based DCFH-DA assay given as percent fluorescence inhibition versus control cells (MeOH only). N=3 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……………………………………….. 99

Figure 28. The photograph shows a) the apparatus used for the photoinhibition assay, and b) the Diving PAM fluorometer………………………….. 105

Figure 29. N=3 ± standard deviation. Different letters indicate significantly different means (P <0.05), Tukey’s HSD……………………………... 106

Figure 30. Morphological changes during the apoptotic death of sympathetic neurons deprived of NGF…………………………………………...... 115

Figure 31. Treatment with 40µM pseudopterosin K, K 2’ac, and K 4’ac significantly enhanced the survival of NGF-deprived mouse sympathetic neuron, N=2 wells ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……… 117

Figure 32. Quantification of ROS in NGF deprived mouse sympathetic neurons treated with pseudopterosins. Different letters indicate significantly different means (P <0.01), Tukey’s HSD……………………………... 119

Figure 33. Confocal micrographs of sympathetic neurons exposed to the dye MitoSox Red a) NGF- (negative control) b) NGF + (positive control) c) ps K 40µM d) ps K 4’ acetate 40µM……………………………….. 120

Figure A1. Photographs of specimens of species in the genus Pseudopterogorgia used in the experiments presented in this dissertation. a) P elisabethae b) P bipinnata, c) P kallos, d) P rigida e) P acerosa, f) P hystrix, g) P spp……………………………………………………………………... 169

1 Figure A2. H NMR spectrum of pseudopterosin A (17) in CD3OD (500 MHz)…. 170

1 Figure A3. H NMR spectrum of pseudopterosin B (18) in CDCl3 (500 MHz)…... 171

1 Figure A4. H NMR spectrum of pseudopterosin C (19) in CDCl3 (500 MHz)…... 172

1 Figure A5. H NMR spectrum of pseudopterosin D (20) in CDCl3 (500 MHz)…... 173

xii 1 Figure A6. H NMR spectrum of pseudopterosin G (27) in CDCl3 (500 MHz).. 174

1 Figure A7. H NMR spectrum of pseudopterosin I (28) in CDCl3 (500 MHz)… 175

1 Figure A8. H NMR spectrum of pseudopterosin J (29) in CDCl3 (500 MHz)... 176

1 Figure A9. H NMR spectrum of ps A-D aglycone (26) in CDCl3 (500 MHz)... 177

1 Figure A10. H NMR spectrum of ps G-J aglycone (34) in CDCl3 (500 MHz)… 178

1 Figure A11. H NMR spectrum of pseudopterosin K (35) in CDCl3 (500 MHz).. 179

1 Figure A12. H NMR spectrum of 2’Acetate ps K (36) in CDCl3 (500 MHz)….. 180

1 Figure A13. H NMR spectrum of 4’Acetate ps K (37) in CDCl3 (500 MHz)…. 181

1 Figure A14. H NMR spectrum of iso-ps A (30) in CD3OD (500 MHz)………... 182

1 Figure A15. H NMR spectrum of iso-ps C (32) in CDCl3 (500 MHz)…………. 183

1 Figure A16. H NMR spectrum of iso-ps D (33) in CDCl3 (500 MHz)…………. 184

xiii

CHAPTER 1

Introduction and Background

1.1-Coral reefs and coral bleaching

Corals and all of the associated biodiversity on reefs are being degraded at an alarming rate. Among the most degraded reefs are those of the Caribbean, which have

suffered an 80% decline in coral coverage over the last thirty years (Gardner et al., 2003) and a consequent phase shift from hard coral to algal dominance (e.g. Hughes, 1994).

Acropora palmata and cervicornis once spatially dominant and the primary

framework builders of the Caribbean during the Pleistocene and Holocene (Carpenter et

al., 2008) are now officially listed as threatened under the U.S. Endangered Species Act,

while macroalgal cover has risen in the last thirty years from an average of ~ 5% to 40%

(Jackson, 2008). This transition has been the result of major disturbances to the

ecosystem including altered food webs (Jackson 1997; Jackson et al. 2001), large scale

bleaching events (Hoegh-Guldberg 1999; Wilkinson 2002), and the emergence of disease

(Harvell et al. 1999; Harvell et al. 2002; Sutherland 2004). Several factors may have

contributed to this process, including over-fishing, increasing CO2 levels, changes in sea

surface temperatures, eutrophication, sedimentation, and pollutants. However, the

individual and synergisitic role of these stressors in causing reef decline is unclear.

Ecosystems are composed of biological communities and habitats. Biological

communities are interacting populations of individual species. Coral reef ecosystems

include the species residing in these habitat types, but they also include aquatic residents

1 of associated sand, macroalgae, seagrass, and mangrove habitats. Species have

specialized roles or niches within habitats. Organisms within coral reef communities can

be divided into four main groups. First, the epibenthos (sessile organisms, the living

substrate) provides the complex structure of the reef itself. These are the coralline and

fleshy algae, hard and soft corals, and sponges. Second, (tiny floating plants and

, most microscopic) provides food for the reef filter feeders. Third, the

suprabenthos are the larger mobile animals that swim over and around the reef. These are the herbivores, corallivores, carnivores and detritivores. Fourth, the cryptofauna, which bores into the substrate and settles in holes and reef crevices. These bryozoans, sponges, tunicates, and polychaetes further increase reef habitat complexity.

Coral reefs form preferentially on elevated stable substrates and within a narrow range of physical parameters. They are restricted to depths of 0-50 m (within the photic zone), salinities between 32 and 38 ppt, temperatures that range between 22 and 29º C

(mean annual values), light levels of 100-2000 µ E m-2 s-1, waters with transmittance

values < 90% and near 90% full saturation oxygen levels. As a result of these

requirements, coral reefs are restricted to the tropics, primarily between 25º north and

south latitude, and predominantly on the western boundaries of the world’s oceans in the

Caribbean and the Indo-Pacific (Porter and Tougas, 2001).

Corals are ancient animals that evolved into the modern reef-building forms over

the last 250 million years. They initially appeared as solitary forms in fossils more than

400 million years ago (Romano and Palumbi, 1996; Romano and Cairns, 2000; Stanley,

2003). The primary building blocks of a reef are polyps of scleractinian corals. These

polyps are sessile with a small cylindrical body and prey-capturing tentacles surrounding

2 the opening or mouth. The polyps of scleractinian corals deposit a calcium carbonate

skeletal cup around themselves. A coral reef is comprised of millions of these calcified

polyps making up individual coral heads. Coral heads are often cemented together by

coralline (calcareous) algae. Symbiotic photosynthetic, single-celled algae of the genus

Symbiodinium, (collectively called zooxanthellae) live in the tissues of each coral .

Polyps depend on these microscopic plants for part of their nutrition, so are limited by their symbionts’ requirements to a maximum depth light penetrates in clear, oceanic

waters (around 150 ft). Corals also rely on intense feeding by herbivorous fishes and sea

urchins to remove seaweeds that can overgrow and kill corals. Processes such as

overfishing of herbivores, climate change, nutrient pollution, sedimentation, and diseases

destabilize these mutualisms and degrade reefs. In 1983 and 1984, the dominant

herbivore of most Caribbean reefs, the long-spined urchin Diadema antillarum, suffered

a pathogen-induced mass mortality. Within a year, >90% of the population of this species had died and algal biomass increased rapidly throughout Caribbean reefs (Hughes, 1994).

Reefs also rely on nearby ecosystems such as seagrass beds and mangrove forests as important sources of energy and nutrients and to provide nurseries for juvenile fishes that

move to reefs as adults. These have suffered unprecedented losses due to coastal

development and associated run-off.

Coral reef ecosystems have evolved in oligotrophic waters with a relative absence of

dissolved nutrients (nitrogen and phosphorus). Yet reefs have rates of carbon fixation that rank them among the most productive ecosystems on Earth (Grigg et al., 1984). The coral reef is the most complex, species-rich, and productive marine ecosystem (Bryant et al.,

1998; Stafford-Deitsch, 1993). It has been estimated that coral reefs have about 1 million

3 species, with only 10% percent described (Reaka-Kudla, 1996). The reefs are home to the

greatest phyletic diversity on the planet, with 32 of the 34 describe phyla found in this

ecosystem (Porter and Tougas, 2001). Reef-building corals form a complex topography

and create a very stable substrate, both of which may persist even after the death of the

coral. This topographic relief and spatial heterogeneity along with the cryptic refuges

they provide facilitate the existence of an abundance of specialized interactions and an

enormous diversity of ecological niches.

The endodermal tissue lining the gastrovascular cavity of scleractinian corals harbors dinoflagellate zooxanthellae (genus Symbiodinium). These endosymbiotic algae play a pivotal role in the survival of the coral host since they provide their host with energy

from photosynthate, primarily in the form of glycerol, but also as glycerides, fatty acids, lipids, organic acids, sugars and amino acids (Muscatine et al., 1972; Muscatine and

Porter, 1977; Patton et al., 1977; Tiltyanov and Tiltyanova, 2002). Zooxanthellae may translocate as much as 95% of their photosynthetic products to the host (Muscatine,

1990). In return, the host provides the zooxanthellae with by-product of respiration such as CO2, organic and inorganic nitrogen and phosphorus. Additionally they receive

protection against herbivory. The most significant and apparently limiting nutrient in the

picture is fixed nitrogen, a critical element in the construction of all proteins. The

symbiotic arrangement allows the partners to avoid the loss of fixed N to the water, a

process that normally occurs in both free floating algae and solo-living marine life

forms. Symbiotic zooxanthellae provide their hosts with orders of magnitude more

energy than normally available to heterotrophic organisms. This is accomplished through

the production of a photosynthate consisting of carbohydrates and lipids and the

4 intracellular translocation of this photosynthate to the cells of the coral host. This

energetic advantage is really what permits zooxanthellate corals to prosper in nutrient-

limiting environments that otherwise could not support them (Stanley, 2003).

The critical coral-algal symbiotic relationship is highly sensitive to environmental changes, particularly during periods of elevated sea surface temperature. This plant- animal partnership comes at a metabolic cost since photosynthetic oxygen released by

algal symbionts can greatly exceed the respiratory demand of the animal (Richier et el.,

2005). This causes coral tissues to be hyperoxic (>250% air saturation) during daylight

exposure which, in combination with high light intensities, can cause photooxidative

toxicity to the symbiosis via the photodynamic production of cytotoxic ROS (Richier et

el., 2005). Coral bleaching occurs when either there is an overall decline in the densities

of zooxanthellae and/or the concentration of photosynthetic pigments within the

zooxanthellae fall (Kleppel et al. 1989). Healthy reef-building corals contain around 0.5-

5 x 106 zooxanthellae cm-2 of live surface tissue and healthy zooxanthellae contain

between 2-10 pg of chlorophyll. When corals bleach they commonly lose 60-90% of their

zooxanthellae and each zooxanthella may lose 50-80% of its photosynthetic pigments

(Glynn 1996). The combination of higher than average temperatures (as small as 1°C–

2°C) and intense light can lead to mass coral bleaching events, which result in apoptosis

and/or the expulsion of the zooxanthellae from the host tissue. Of great debate is the

adaptive bleaching hypothesis, which views bleaching as having an adaptive value by

allowing the host to rid itself of its resident phylotype of algae in order to increase the

chances of becoming repopulated with a different phylotype of zooxanthellae that may be

more tolerant of the new environmental regime (Buddemeier and Fautin, 1993).

5 In the early to mid 1980’s coral reefs around the world began to experience large

scale bleaching (Brown, 1997; Glynn, 1996; Goreau, 1992; Glynn, 1991; Hayes and

Goreau, 1991; Goenaga and Canals, 1990; Goreau, 1990; Jokiel and Coles, 1990;

Williams and Bunkley-Williams, 1990). Since then, bleaching events have occured

almost every year, at one time or another impacting every reef region of the world, and across all depths of the tropical reef (Hoegh-Guldberg, 2005). The severity of these

events has ranged from catastrophic, resulting in mass mortality throughout at least

eighty percent of a reef system, to negligible in cases where only small patches of reef

bleach and then recover (Wilkinson et al., 1999; Wilkinson, 1998). Factors that may

influence the intensity and outcome of bleaching episodes include differences in

genetically-based species- and population-specific responses (Buddemeier and Fautin,

1993), the location of the reef (e.g., depth), strength of the stressor(s) involved, and the

duration of the stress (Wilkinson, 1998). Recent years have seen widespread and severe

coral bleaching episodes around the world, with coral mortality reaching 70% in some regions. The decline of coral reefs is significant, because coral reefs maintain high levels

of biodiversity, provide habitats for coastal fisheries, may contain potential

pharmacological compounds, and protect shorelines from erosion. Trends of the past

century suggest that coral bleaching events may become more frequent and severe as the

climate continues to warm, exposing coral reefs to an increasingly hostile environment.

This global threat to corals compounds the impacts of more localized anthropogenic

factors that already place reefs at risk.

In response to a dramatic increase in the frequency and intensity of bleaching events over the past 15–20 years, research focused on the molecular events occurring

6 during the dissociation of dinoflagellate-cnidarian symbioses have intensified. Bleaching

is considered a symptom of stress and may be induced by a variety of physical and

biological factors, including high and low sea surface temperatures (Jokiel and Coles,

1977; Fitt and Warner, 1995; Saxby et al., 2003; Hoegh-Guldberg and Fine, 2004), UV irradiation (Jokiel and Coles, 1977; Gleason and Wellington, 1993; Brown et al., 1994), bacterial infection (Kushmaro et al., 1996; Rosenberg and Falkovitz, 2004), lowered salinity, and pollution (Glynn, 1993). All of these factors have the ability to cause localized bleaching events; but it is increased SST and irradiance associated with periods of El Nino Southern Oscillation that causes mass bleaching and poses the greatest challenge to coral reefs worldwide (Hoegh-Guldberg and Smith, 1989; Porter et al., 1989;

Glynn, 1991 and Glynn, 1993; Fitt and Warner, 1995; Hoegh-Guldberg, 1999 and

Hoegh-Guldberg, 2004; Lough, 2000).

During conditions of high light and temperature, over-reduction of the electron transport chain downstream of the Photosystem II (PS II) reaction centre (Jones and

Hoegh-Guldberg, 2001) contributes to an increased production of reactive oxygen species

(ROS) within the dinoflagellates (Lesser,1997). Chloroplasts are hyperoxic, produce

ROS, and are susceptible to oxidative stress. ROS in the chloroplast may damage PS II,

primarily through oxidative degradation of the D1 protein (Warner et al., 1999), and also

1 − inhibit the repair of damage to PS II (Nishiyama et al, 2001). In addition to O2, O2 and

HO• also are produced in the PS II reaction center.

Oxidative stress—the production and accumulation of reduced oxygen

intermediates such as superoxide radicals, singlet oxygen, hydrogen peroxide, and

hydroxyl radicals—can damage lipids, proteins, and DNA. Many disease processes and

7 the aging process involve oxidative stress in their underlying etiology. The production of

reactive oxygen species is also prevalent in the world’s oceans, and oxidative stress is an

important component of the stress response in marine organisms exposed to a variety of

insults as a result of changes in environmental conditions such as thermal stress, exposure

to ultraviolet radiation, or exposure to pollution (Lesser, 2006).

Atmospheric O2 in its ground state is distinctive among the elements because it

has two unpaired electrons (and thus is known as a biradical). This property significantly limits its ability to interact with organic molecules unless it is “activated.” The univalent reduction of molecular oxygen produces reactive intermediates such as the superoxide

− 1 radical (O2 ), singlet oxygen ( O2), hydrogen peroxide (H2O2), and the hydroxyl radical

(HO•). Cumulatively the reduction products of oxygen are known as free radicals or

reactive oxygen species (ROS) (Cadenas, 1989).

− All photosynthetic and respiring cells produce ROS, including O2 via the

− univalent pathway; H2O2 is formed by the continued reduction of O2 ; and eventually

HO• is formed and then reduced to the hydroxyl ion and water. Oxidative stress can be

defined as the production and accumulation of ROS beyond the capacity of an organism

to quench these reactive species. It can result in damage to lipids, proteins, and DNA, but

ROS can also act in signal transduction (Fridovich, 1998). The central purposes of

1 antioxidant defenses in biological systems are to quench O2 at the site of production and

− to quench or reduce the flux of reduced oxygen intermediates such as O2 and H2O2 to prevent the production of HO•, the most damaging of the ROS.

− The reducing side of PS I can reduce O2 to O2 by the Mehler reaction and is the

− most significant site of O2 production in the chloroplast (Lesser, 2006). The production

8 − of O2 increases under stressful conditions, such as exposure to xenobiotics or pollutants, high visible irradiances, exposure to ultraviolet radiation (UVR), and/or exposure to thermal stress. This elevated production can overwhelm antioxidant defenses and can result in damage to both PS II and the carbon fixation process.

The extent to which hosts lose their populations of symbiotic dinoflagellates varies among species (Hoegh-Guldberg and Salvat, 1995; Fabricius, 1999; Loya et al.,

2001; Douglas, 2003), within species (Lasker et al., 1984; Porter et al., 1989; Glynn,

1990; Berkelmans and Oliver, 1999; Ulstrup et al., 2006; LaJeunesse et al., 2007) and sometimes between regions of a colony (Rowan and Knowlton, 1995; Rowan et al.,

1997). This variability has been attributed to the thermal tolerance of the symbiont

(Berkelmans and van Oppen, 2006; Ulstrup et al., 2006), differences in host tolerance

(Loya et al., 2001; Brown et al., 2002b; Bhagooli and Hidaka, 2004; D’Croz and Maté,

2004), or particular host– symbiont combinations (Rowan, 2004; Goulet et al., 2005). In the Caribbean, octocorals are the most conspicuous reef macrofauna, with up to 40 species co-occurring in a single area (Sánchez et al., 2003). A striking difference between

Caribbean octocorals and other zooxanthellate cnidarians is their seemingly greater resistance of bleaching. While there have been numerous reports of scleractinian coral bleaching throughout the world’s tropical oceans, there have been far fewer observations of bleaching among octocorals (Lasker, 2003). If the increasing incidence of bleaching events continues, the relative insensitivity of most gorgonians to high temperatures may have important consequences for Caribbean reefs.

Octocorals or soft corals are important constituents of coral reef ecosystems

(Goldberg, 1973; Kinzie, 1973; Kinzie, 1974; Lasker and Coffroth, 1983; Sánchez et al.,

9 1997; Fabricius and Alderslade, 2001; Sánchez et al., 2003). Few studies, however, have

examined how they and their symbionts differentially respond to environmental stress

(Fabricius, 1999; Strychar et al., 2005). In a study done during the height of the 1998

mass bleaching event on the , the octocoral families Briareidae,

Clavularidae, Gorgonidae, and Nephtheidae exhibited low bleaching susceptibilities

(Goulet et el., 2008). There was no indication that different Symbiodinium populations explained the occurrence of bleached or unbleached tissues. The authors of this study concluded that differential tolerance to stress among cnidarian species, irrespective of the

Symbiodinium they host, may explain differences in bleaching. In the Caribbean, the vast majority of octocorals and other invertebrate hosts associate with Symbiodinium clade B,

and more selectively, with a single lineage of this clade, Symbiodinium B1/B184 (Santos

et al., 2004). Since specificity and stability in cnidarian–dinoflagellate symbioses are

common (Rodriguez- Lanetty et al., 2003, 2004; LaJeunesse et al., 2004; Goulet, 2006;

Thornhill et al., 2006), especially among octocorals (Goulet and Coffroth, 2003, 2004;

Santos et al., 2003; Kirk et al., 2005; Goulet et al., 2008), rapid environmental changes

may not lead to new symbiotic combinations in alcyonaceans. The physiological

resiliency of host and symbiont under their current range of partner combinations may

limit their potential for surviving acute environmental stress.

1.2-Natural products

Humans have made use of natural products throughout most of its history. Plant

extracts have been incorporated in the preparation of foodstuff, dyes, toxins and medicines by even the most ancient human civilizations. In the late eighteenth century,

10 chemists moved this practice into the realm of modern science. They began to separate,

purify and analyze active compounds made inside living cells. Aspirin or acetylsalicylic

acid is a derivative of salicylic acid that is a mild, non-narcotic analgesic useful in the

relief of headache and muscle and joint aches. The drug works by inhibiting the

production of prostaglandins, metabolites that are necessary for blood clotting and which

also sensitize nerve endings to pain.

Hippocrates, who lived sometime between 460 B.C and 377 B.C, is considered

the father of modern medicinal science. Hippocrates has left historical records of pain

relief treatments, including the use of a powder made from the bark and leaves of the

willow tree to help heal headaches, pains and fevers. In the early 1800’s, scientists

discovered that it was the compound called salicin in willow plants that relieves pain. In

1828, Johann Buchner, professor of pharmacy at the University of Munich, isolated a tiny

amount of bitter tasting yellow, needle-like crystals, which he called salicin. In 1838,

Raffaele Piria a chemist at the Sorbonne in Paris, split salicin into a sugar and an

aromatic component (salicylaldehyde) and converted the latter, by hydrolysis and

oxidation, to a crystallized acid, which he named salicylic acid (1). The problem was that

salicylic acid caused stomach irritation. In 1853, a French chemist Charles Frederic

Gerhardt neutralized salicylic acid by buffering it with sodium (sodium salicylate) and

acetyl chloride, creating acetylsalicylic acid (2). In 1899, a German chemist named Felix

Hoffmann, who worked for a German company called Bayer, rediscovered Gerhardt's formula. Felix Hoffmann gave acetylsalicylic acid to his father who was suffering from the pain of arthritis. With good results, Felix Hoffmann then convinced Bayer to market

the new wonder drug. Aspirin was patented on March 6, 1889. The scientists at Bayer

11 came up with the name Aspirin, it comes from the 'A" in acetyl chloride, the "spir" in

Spiraea ulmaria (the plant they derived the salicylic acid from) and the 'in' was a then familiar name ending for medicines (Andermann, 1996).

O O

OH OH

OH O 1 2

O

Figure 1. Structure of salicylic acid (1) and acetylsalicylic acid (2).

A natural product is generally defined as a secondary metabolite; one that is not necessary to sustain the life of the producing organism, but confers to it an adaptive advantage. Secondary metabolites produced by living organisms presumably have adaptive value that favors their evolution. Many plants and animals, both terrestrial and marine, produce compounds that help them survive by, for example, deterring potential predators, warding off pathogens, keeping their living space free from competitors, and reducing the impact of exposure to environmental stresses, such as high levels of UV radiation.

Nature has provided mankind with a broad array of structurally diverse and often pharmacologically active compounds that might be of use to combat disease or as lead structures for the development of novel synthetically derived drugs that approximate their structure and function in nature. Traditionally, higher plants and, since the discovery of the penicillins, terrestrial microorganisms have proven to be the richest sources of novel

12 bioactive compounds. A well-known example of a plant-derived anti-cancer drug is

paclitaxel (taxol®, 3), isolated from the bark of the Pacific yew Taxus brevifolia.

Examples of anti-cancer agents from bacterial sources include doxorubicin (adriamycin,

4) and bleomycin from various Streptomyces strains.

O O AcO HO

NH O

O O H AcO HO OH O

3

O O

OH

O O OH O O

NH2

4 OH

Figure 2. Structures of paclitaxel (3) and doxorubicin (4).

1.3-Marine natural products

The marine environment covers a wide thermal range with temperatures from below the freezing point in Antarctic waters to as high as 350°C in deep hydrothermal

13 vents. The ocean environment also exhibits a vast pressure range (1-1000 atm), nutrient range (oligotrophic to eutrophic) and varied light regimes (photic and non-photic zones).

Due to this extensive variability the ocean is home to unmatched speciation at all phylogenetic levels, from microorganisms to mammals. Despite the fact that the biodiversity of higher taxa in the marine environment far exceeds that of the terrestrial environment, research of marine natural products is still in its infancy. The development of new diving techniques, manned submersibles and remotely operated vehicles and improved technologies for mapping has increased our capacity to collect marine samples.

Exploration of the vast potential of marine organisms as sources of bioactive metabolites started in the late 1960s. The discovery of prostaglandins (important mediators involved in inflammatory diseases, fever and pain) in the gorgonian Plexaura homomalla by Weinheimer and Spraggins in 1969 is considered to be a critical starting point for the search for “drugs from the sea” (Weinheimer and Spraggins, 1969). From

1969–1999 approximately 300 patents on bioactive marine natural products were issued.

The number of compounds isolated from various marine organisms now exceeds 10,000

(MarinLit, 2001). There are currently three FDA approved drugs of marine origin.

Unusual nucleosides isolated from the Caribbean sponge Cryptotethya crypta (Bergmann and Feeney, 1951) served as lead structures for the development of the now commercially important anti-viral drug ara-A (5) and the anticancer drug for leukemia ara-C (6) (Arif et al., 2004). Ziconotide (Prialt®, 7), a 25 amino acid peptide isolated from the marine mollusk Conus magnus, was approved by the FDA in 2004 for use in alleviating chronic pain (Proksch et al., 2002; Associated Press, 2004; Newman et al., 2004).

14 NH2 NH2 N N N

N OH N O N O OH OH O OH OH 5 6 OH

H2N CKGKGAKCSRLMYDCCTGSCRSGKC CONH2 7

Figure 3. structures of ara-A (5), ara-C (6) and Prialt® (7)

Many marine natural products are currently in clinical trials. Notable anti-cancer

compounds from marine organisms include bryostatin 1 (8) and ecteinascidin-743 (9)

(Yondelis®). Bryostatin 1 is polyketide isolated from the bryozoan Bugula neritina and is currently in Phase I/II clinical trials for treatment of various cancers (Newman et al.,

2004). Currently there are four phase I and five phase II trials underway for the use of bryostatin 1 in combination with nucleotide derivatives or cytotoxic agents (Newman et al., 2004). Ecteinascidin-743 is a tetrahydroisoquinoline alkaloid from the tunicate

Ecteinascidia turbinata. It is presently in phase III clinical trials and is effective against solid tumors such as sarcomas and breast cancer (Proksch et al., 2002; Arif et al., 2004).

15 O O H OH HO OAC MeOOC O H H O O N H S H O N OH OH O H O OHO O OH O O O HO O NH O COOMe HO 8 9

Figure 4. Structures of bryostatin 1 (8) and ecteinascidin-743 (9).

Marine octocorals (Phylum ) are a source of a wealth of biologically active and structurally unique compounds isolated and described by natural products chemists (Faulkner, 2000). These metabolites have been assumed to be important for the success of octocorals in the marine environment, and their real ecological functions have been experimentally tested (Cronin et al., 1995; Fenical and Pawlik, 1991; Harvell et al.,

1988; Pawlik and Fenical, 1992; Pawlik et al., 1987; O’Neal and Pawlik, 2002). The defensive role against has been the most analyzed, in part due to the low predation rates found in these sessile and soft-bodied animals. Living in habitats characterized by high levels of predation and nutrient scarcity, octocorals seem to be free from predation with the exception of certain specialists such as the mollusks Cyphoma gibbosum (Harvell and Suchanek, 1987), and the butterfly fish Chaetodon capistratus

(Lasker, 1985).

Caribbean gorgonians account for nearly 38% of known octocorals and over 195 species of octocorals have been recorded from this region (Fenical, 1987). Gorgonians

16 include organisms commonly referred to as sea plumes, sea rods, flat sea whips, and sea fans. Gorgonians have a central axis that is attached to a substrate by a holdfast.

Radiating from the central axis are branchlets that contain polyps, which extend themselves through apertures called calyces for feeding. The shapes of these calyces are taxonomically distinct (Bayer, 1961). Different branching patterns and the three dimensional arrangement of these branching patterns also define different genera and species (Bayer, 1961).

Evidence of chemical defense among gorgonians is abundant. While detailed chemical analysis has only been conducted on about one-fifth of known Caribbean gorgonian species, a large number of novel metabolites have been discovered in the tissue of these organisms (reviewed in Rodriguez, 1995). Many natural products isolated from gorgonians are active in pharmacological assays, many exhibiting anti-cancer and anti- inflammatory properties (Fenical, 1987; Rodriguez, 1995). Hypotheses regarding the ecological role of secondary metabolites have been tested in some cases. There is experimental evidence that the compounds may inhibit larval settlement, increase resistance to fungal pathogens, or prevent or inhibit overgrowth by other sessile organisms (Standing et al., 1984; Rodriguez, 1995; Kim et al., 2000). The most common hypothesis is that secondary metabolites are anti-predatory agents, and studies have consistently demonstrated antipredatory properties of gorgonian crude extracts and purified compounds (Pawlik et al. 1987; Fenical & Pawlik, 1991; Pawlik and Fenical,

1992; Paul and Van Alstyne, 1992; Harvell et al., 1993; Epifanio et al., 1999; Maia et al.,

1999; Koh et al. 2000).

17 The wealth of bioactive metabolites isolated from soft-bodied, sessile or slow-

moving marine invertebrates that lack morphological defense structures such as spines or a protective shell is no coincidence but reflects the ecological importance of these coumpounds for these organisms. It has been shown that chemical defense through accumulation of toxic or distasteful natural products is an effective strategy to fight off potential predators and/or organisms competing for space (Proksch et al., 2002;

McClintock and Baker, 2001). Grazing pressure by predators such as fishes is higher on

tropical and subtropical reefs than in any other ecosystem of the world. On tropical coral

reefs fish have been estimated to bite the bottom in excess of 150,000 times per m2 per day (Carpenter, 1986). With this intense selective pressures survival depends on effective means of chemical defense.

In many cases compounds that protect their invertebrate producers from predators or that help to fight off fouling organisms and those organisms competing for space have also attracted attention in pharmacological assays that are aimed at drug discovery. Thus, organisms that thrive in spite of pronounced biotic pressures can, to some degree, be expected to contain metabolites that are also of interest for drug discovery programs.

While there is no direct correlation between defensive and biomedical potential of a

compound, the fact that many cellular processes and structures such as enzymes and

receptors are highly conserved implies that they must have been good targets for inter-

species chemical warfare. For example, the production of a substance by one organism in

order to affect a second organism in a detrimental manner- the broadest definition of

antibiosis- is very common phenomenon in the marine ecosphere and may provide a

convincing rationale for the production of antimicrobial and antiviral agents by marine

18 invertebrates that are constantly in contact with marine bacteria, fungi and viruses. The

rich diversity in chemical structures found in marine phyla may reflect the long period of time the organisms have had to perfect their chemical arsenal (Reviewed in Faulkner,

2000).

Gorgonian corals are found in all tropical and subtropical seas, but reach their

greatest levels of diversity and abundance on Caribbean coral reefs (Bayer 1961, Kinzie

2 1973), where they attain densities as high as 25 colonies per m (Goldberg 1973).

Faunistically, extensions of the West Indian region reach into the Gulf of Mexico, all the

Antilles, the Bahamas, the Florida Keys, the Bermudas, the Islands of the Caribbean, and south along the northeast coast of South America to the reefs of Brazil. All over this

region, the families and Plexauddae flourish as they do nowhere else in the

world. Gorgonian corals (order Gorgonacea, phylum Cnidaria) are conspicuous members

of most tropical and subtropical marine habitats, being the most abundant octocorals

found in the West Indies. With over 195 species documented from these two major

families, gorgonian octocorals represent an estimated 38% of the known fauna.

Taxonomically, the include six orders the (fleshy soft

corals), Telestacea (common fouling organisms), Gorgonacea (horny or gorgonian soft

corals), Stolonifera (matlike or stolonborne soft corals), Pennatulacea (sea pens), and

Coenothecalea (blue-stony corals). Defensive chemistry has been reported or noted from all six orders (Coll, 1992). The Alcyonacea and Stolonifera are prominent in the Indo-

Pacific, while the Gorgonacea and Pennatulacea dominate the Caribbean. There is little crossover of species between the two regions.

19 Unlike scleractinian corals, which have only a thin veneer of living tissue on an

endoskeleton of rock-hard carbonate, gorgonians possess a relatively fleshy coenenchyme

that usually surrounds a supportive rod or mesh of sclerotinized protein. Given the

nutrient scarcity and high levels of predation reported for coral reef environments (Grigg et al., 1984), it might be expected that gorgonians would provide a source of food for a variety of reef predators, but this does not appear to be the case (Pawlik et al., 1987).

Natural products chemists have isolated over 200 novel secondary metabolites from these animals (Faulkner, 2000; and previous reviews by the same author); some of these compounds occur at extraordinarily high concentrations within the coral tissues

(Schneider et al., 1977). Although pharmacological effects of many of these compounds have been described (see references in Pawlik et al., 1987), only recently have the

possible functions of these metabolites in gorgonians been addressed by employing ecologically relevant laboratory and field experiments (Gerhart, 1988; Pawlik et al.,

1987; Harvell et al., 1988; Harvell and Fenical, 1989; Pawlik and Fenical, 1989).

Gorgonians of the genus Pseudopterogorgia are commonly referred to as "sea plumes" based on their finely branch or plumose growth forms. Pseudopterogorgia

species are among the most common of the Caribbean species. The first chemical

analysis of a Pseudopterogorgia species began in 1968, with investigations of the

sesquiterpene hydrocarbons from Pseudopterogorgia americana (Schmitz et al., 1969).

The gorgonian corals, and octocorals in general, are a rich source of biologically active

compounds which play a defensive role against predators (e.g. Pawlik et al. 1987; Harvell

et al. 1988; Harvell and Fenical, 1989; Pawlik and Fenical, 1989; Fenical and Pawlik,

1991; Paul and Van Alstyne, 1992; Van Alstyne et al. 1992), competitors (Sammarco et

20 al.,1983; La Barre et al., 1986), and microbes and fouling organisms (Targett et al., 1983;

Bandurraga and Fenical, 1984; Gerhart et al., 1988; Ciereszko and Guillard, 1989; Kim,

1994; Slattery et al., 1995; Jensen et al., 1996).

Chemical defenses isolated from gorgonians and alcyonaceans are usually terpenoids (reviewed in Paul and Puglisi, 2004). Briarane and asbestinane diterpenes are found in adult colonies and the larvae of the gorgonian Briareum asbestinum and show anti-predator activity in fish feeding assays (Harvell et al., 1996). Similarly, pukalide and

11-acetoxypukalide were found in the eggs of the Pacific soft coral Sinularia polydactyla

(Slattery et al., 1999). The concentration of pukalide was similar to that of the adults while the concentration of 11-acetoxypukalide was much lower. Recently, the sesquiterpene heterogorgiolide and a known eunicellane diterpenoid from Heterogorgia uatumani were reported to be unpalatable to natural assemblages of fishes in Brazil (Maia et al., 1999). A similar study of the gorgonian Lophogorgia violacea yielded a mixture of

5 furanocembranoid diterpenes that in combination also appear to deter fish predation

(Epifanio et al., 2000).

Antimicrobial activities of crude extracts from many Caribbean gorgonian corals have been examined (reviewed in Paul and Puglisi, 2004). Bioassays carried out with the extracts of seven Caribbean gorgonians in the family and one gorgonian in the family Gorgonidae showed that non-polar fractions inhibited the growth of three species of marine bacteria and two non-marine species (Kim, 1998). Among cnidarians, gorgonians display some of the most potent antimicrobial activities (Jensen et al. 1996;

Kim et al., 2000). The hydroquinones of Pseudopterogorgia rigida and P. acerosa have antiviral and antibacterial activity and deter predatory fish (Harvell et al., 1988). In a

21 broad survey of 39 Caribbean sea fans and sea whips, crude extracts were screened for antibacterial activity by the standard agar disc-diffusion method against a host of marine bacteria isolated from the surfaces of living colonies of Briareum asbestinum and decaying gorgonians and three bacterial species known to be pathogenic to marine invertebrates (Jensen et al., 1996). While only 15% of the extracts showed antimicrobial activity, extracts from Pseudopterogorgia spp. were quite active, inhibiting the growth of most bacterial strains tested.

Gorgonians from the Caribbean region are responsible for several diterpenoid carbon skeletal classes including cembranes, asbestinins, briareins, pseudopterosins, and pseudopteranes, along with other skeletons that do not belong to any major class. Most of these skeletal classes possess unique functionalities and substitution patterns.

Cembranes represent the largest group of compounds isolated from gorgongians in the

West Indian region (Rodríguez, 1995).

The gorgonian Pseudopterogorgia bipinnata, a widely distributed member of the genus Pseudopterogorgia, produces a large variety of cembrene derivatives somewhat dependent upon collection site. The characteristic of Pseudopterogorgia cembrenes is their high levels of oxygenation. Bipinnatins A-D (10-13) were first isolated from P. bipinnata collected off Jamaica Cay and Acklins Islands, Bahamas (Wright et al., 1989).

Bipinnatins A, B, and D were active, in vitro, against P388 murine tumor cell lines with

IC50 values 0.9, 3.2, and 1.5 µg/mL, respectively. Additionally, bipinnatin B was found to inhibit [125I]-α-toxin binding to intact cells and block α-toxin binding to detergent- extracted receptor. Bipinnatin B also inhibits [125I]-α-toxin binding to receptor-rich

22 membrane fragments prepared from Torpedo electric organ (Culver et al., 1985,

Abramson et al., 1991).

OAc OAc O R O O O O CHO

OAc OAc O O O O 13 O R= CO2CH3 CHO 10 12 11

Figure 5. Structures of bipinnatans A-D (10-13).

Some P. bipinnata also contain diterpenes representative of the pseudopterane skeletal class including, kallolide A (14) and kallolide A acetate (15) (Look et al, 1985).

Kallolide A has been shown to be a potent inhibitor of phorbol ester induced inflammation in the mouse ear assay at concentrations and efficacies equivalent to those of indomethacin (Marshall and Nelson, 1988).

OR

O

O

14 R=H 15 R=Ac O

Figure 6. Structures of kallolide A (14) and kallolide A acetate (15).

23 The pseudopterosins are a class of structurally diverse anti-inflammatory and

analgesic metabolites isolated from the gorgonian Pseudopterogorgia elisabethae (Look

et al., 1986). The pseudopterosins show anti-inflammatory activity equal to or great than

the industry standard, indomethacin, and appear to possess a unique mechanism of action.

There is also recent evidence suggesting that pseudopterosins may serve as anti-oxidants

(Mydlarz and Jacobs, 2004). Currently pseudopterosins are used as cosmetic additives

(Rouhi, 1995).

Pseudopterosins A-D (17-20) were originally from samples collected at Crooked

Island, Bahamas (Look et al., 1986). The structure of pseudopterosin C was established

by X-ray crystallography. P. elisabethae collected from different geographic locations

posses different congeners of the pseudopterosins. So far, twenty-six pseudopterosin

congeners have been described, all exhibiting the amphilectane skeleton and a glycoside

linkage at either C-9 or C-10 (Rodriguez, 1995).

H O O OR3 R1O OR2 OH

17 R1= R2= R3=H 18 R1= Ac, R2= R3=H 19 R2= Ac, R1= R3=H 20 R3= Ac, R1= R2=H

Figure 7. Structures of pseudopterosins A-D (17-20)

Due to their excellent antiinflammatory and analgesic activity, partially purified extracts containing pseudopterosins are currently incorporated into skin care preparations

24 (Pomponi, 1999). The availability of pseudopterosins however, is limited by the actual supply of organic extracts of Pseudopterogorgia elisabethae which currently only comes from the Bahamas islands. The complex and expensive chemical synthesis of these compounds makes these animals an attractive study target in other areas of the Caribbean.

One of the arguments often advanced for the conservation of coral reef ecosystems is the extraordinary diversity of these communities and the potentially useful bioactive compounds that reef species may produce (Bruckner, 2002). That potential creates a paradox, in that the discovery of compounds with potentially valuable properties generates the need to harvest that material. Even when laboratory syntheses of the compounds are possible it is often the case that the amounts of material needed for exploratory research will necessitate extensive collections. Bruckner (2002) for instance notes cases in which thousands of kilograms of tissue are needed to generate milligrams of the target compound.

To date, pseudopterosins from the Caribbean octocoral Pseudopterogorgia elisabethae are the only compounds from a reef species that have made their way to commercial use. Pseudopterosins exhibit anti-inflammatory and analgesic properties and are used by the cosmetics industry (Look et al., 1986). Over the last 12 years P. elisabethae has been harvested from the Little Bahama Bank in the Bahamas. The fishery is regulated through an export limit set by the Bahamas Department of Marine Resources.

Annual harvest data are not publicly available, but Bruckner (2002) placed the value of the fishery at $3–$4 million and Puyana et al. (2004) notes that estimated demand for the pseudopterosins is 13–20 tons per year. The collection protocols in the fishery have been developed by the collectors. Colonies are collected by divers using either scuba or surface

25 supplied air. The collectors clip the colony, removing most of the colony but with the

goal of leaving at least one central branch, typically 5–15 cm in height, that contains 5–

10 branchlets. The remaining tissue survives and regenerates new branches (Castanaro

and Lasker, 2003). Colonies develop sufficient tissue for reharvesting in as little as 2

years (Goffredo and Lasker, 2008). Initially populations were allowed to recover for 2–3 years, which was the time generally required for new and recovering colonies to reach a size at which a harvest would be profitable. Additional input from more recent scientific studies (i.e., Lasker et al., 2003, Gutiérrez-Rodríguez and Lasker, 2004) has led the collectors to extend the recovery time to allow colonies to reach reproductive size and reproduce prior to a second harvest. Some of the sites have been harvested 3 times which is suggestive of sustainability, but the fishery has not been the subject of a quantitative analysis.

1.4 Reactive oxygen and antioxidants

Molecular oxygen is relatively unreactive due to its electron configuration.

Activation of oxygen (i.e. the first univalent reduction step) is energy dependent and

requires an electron donation. The subsequent one-electron reduction steps are not energy

dependent and can occur spontaneously or require appropriate e–/H+ donors. In biological

systems transition metal ions (Fe2+, Cu+) and semiquinones can act as e– donors. Four-

electron reduction of oxygen in the respiratory electron transport chain (ETC) is always

accompanied with a partial one- to three-electron reduction, yielding the formation of

ROS (Halliwell and Gutteridge, 1999). This term includes not only free radicals

·– · (superoxide radical, O2 , and hydroxyl radical, OH ), but also molecules such as

26 1 ·– hydrogen peroxide (H2O2), singlet oxygen ( O2) and ozone (O3). Both O2 and the

· hydroperoxyl radical HO2 undergo spontaneous dismutation to produce H2O2. Although

·– 2+ H2O2 is less reactive than O2 , in the presence of reduced transition metals such as Fe in a chelated form (which is the case in biological systems), the formation of OH· can occur in the Fenton reaction (Halliwell and Gutteridge, 1999).

A biological antioxidant has been defined as any substance that when present at low concentration compared with that of an oxidizable substrate significantly delays or prevents oxidation of that substrate (Halliwell and Gutteridge, 1999). Since some free

radical production in cells is inevitable and can be very damaging, a defense system

against the deleterious action of free radicals has evolved (Cheeseman and Slater, 1993).

These are known as antioxidant defenses and the two main categories are those whose

role is to prevent the generation of reactive oxygen species (ROS), and those that

intercept any radicals that are generated (Larson, 1997).

A free radical can be defined as a chemical species possessing an unpaired

electron. It can also be considered as a fragment of a molecule. As such, free radicals can

be formed in three ways: (i) by the hemolytic cleavage of a covalent bond of a normal

molecule, with each fragment retaining one of the paired electrons; (ii) by the loss of a

single electron from a normal molecule; (iii) by the addition of a single electron to a

normal molecule (Halliwell and Gutteridge, 1999). The latter, electron transfer, is a far

more common process in biological systems than is homolytic fission, which generally

requires high energy input from either high temperatures, UV light or ionizing radiation.

Heterolytic fission, in which the electrons of the covalent bond are retained by only one of the fragments of the parent molecule, does not result in free radicals but in ions, which

27 are charged. Free radicals can be positively charged, negatively charged or electrically

neutral. The unpaired electron and the radical nature of a species are conventionally indicated by writing it with a heavy superscript dot.

It can be a source of confusion that the electrons in one of the most important molecules in free radical biochemistry, oxygen, are distributed in such a way that two of the electrons are 'unpaired'. Thus, oxygen is sometimes considered a di-radical. While the di-radical nature of oxygen does enable it to react readily with many other free radicals, in general it reacts relatively slowly with non-radical species (Denisov and Afanas’ev,

2005). When considering its reactions in the context of free radical biochemistry, it is

usually easiest to simply consider it as a normal molecule that can readily add to free

radicals or accept a single electron from them, while not itself being a free radical

(Halliwell and Gutteridge, 1999).

Arguably the most important free radicals in biological systems are radical derivatives of oxygen (Cheeseman and Slater, 1993). Reactive oxidizing species derived from oxygen are formed in cells by normal metabolism as well as by numerous types of environmental insults. Reduction of oxygen by the transfer to it of a single electron will

produce the superoxide free radical anion ('superoxide').

- -· O2 + e —>O2

A two-electron reduction of oxygen would yield hydrogen peroxide:

- + O2 + 2e + 2H

Hydrogen peroxide is often generated in biological systems via the production of

superoxide: two superoxide molecules can react together to form hydrogen peroxide and

oxygen.

28 -· + 2 O2 + 2H —> H2O2 + O2

Because the free radical reactants produce non-radical products this is known as a

dismutation reaction. It can take place spontaneously (slow reaction kinetics) or can be

catalyzed by the enzyme superoxide dismutase. Hydrogen peroxide is not a free radical

but falls into the category of 'reactive oxygen species' (ROS) that includes not only

oxygen free radicals but also non-radical oxygen derivatives that are involved in oxygen

radical production (Denisov and Afanas’ev, 2005).

Hydrogen peroxide is an important compound in free radical biochemistry

because it can rather easily break down, particularly in the presence of transition metal ions, to produce the most reactive and damaging of the oxygen free radicals, the hydroxyl radical (·OH):

2+ · - 3+ H2O2 + Fe —> OH + OH + Fe

The above reaction is referred to as the iron-catalysed Haber-Weiss reaction. The non-

catalysed Haber-Weiss reaction is the reaction of superoxide directly with hydrogen

peroxide:

-· + · - O2 + H2O2 —> OH + OH + O2

All of the major classes of biomolecules may be attacked by free radicals but

lipids are probably the most susceptible. Cell membranes are rich sources of

polyunsaturated fatty acids (PUFAs), which are readily attacked by oxidizing radicals.

The oxidative destruction of PUFAs, known as lipid peroxidation, is particularly damaging because it proceeds as a self-perpetuating chain-reaction. Lipid peroxidation has been implicated in a wide range of tissue injuries and diseases (Romero et al, 1998;

Pansarasa et al., 1999; Beal, 2002).

29 Free radical scavengers are preventive antioxidants. Antioxidants can act at

different levels in an oxidative sequence. This may be illustrated by considering one of

the many mechanisms by which oxidative stress can cause damage by stimulating the

free radical chain reaction of lipid peroxidation (Denisov and Afanas’ev, 2005). Free radical chain reactions within a cell can be inhibited by adding chemicals that retard the formation of free radicals, by introducing substances that compete for the existing radicals and remove them from the reaction medium.

Antioxidative properties of polyphenols arise from their high reactivity as

hydrogen or electron donors, and from the ability of the polyphenol-derived radical to stabilize and delocalize the unpaired electron (chain-breaking function), and from their

ability to chelate transition metal ions causing the termination of the Fenton reaction

(Rice-Evans et al., 1997). Phenols are intrinsically electron-rich compounds prone to enter into efficient electron-donating reactions with oxidizing agents. Typically, an electron is transferred from a phenol to the unfilled orbital of a one-electron oxidant such as a peroxyl radical, followed by rapid proton transfer:

Ph-OH + ROO· → Ph-O· + ROOH

The net result is the equivalent of a hydrogen atom transfer from the phenolic hydroxyl group to the free radical. The product, the phenoxyl radical, PhO·, is stabilized by

resonance delocalization of the unpaired electron to the ortho and para positions of the

ring (Denisov and Afanas’ev, 2005).

30 In order to act as an effective antioxidant, the phenoxyl radical must not be

reactive enough to initiate further free-radical reactions on its own. While the phenol

itself is a rather ineffective antioxidant, if other radical-stabilizing features are

accentuated, extremely potent antioxidants can result (Denisov and Afanas’ev, 2005). In

addition to the intrinsic stability of the resulting radical due to resonance properties, various structural features such as the steric and inductive effects of ring substituents enhance this stability. For example, bulky or electron donating groups at the ortho

position of a phenol is the signature feature of many synthetic and naturally occurring

antioxidants (Denisov and Afanas’ev, 2005).

Diterpenes play diverse functional roles in vivo, acting as hormones

(gibberellins), regulators of wound-induced responses (abietic acid), photosynthetic

pigments (the phytyl chain of chlorophylls), and antioxidants (McGarvey and Croteau,

1995). Although tocopherols and carotenoids are, among lipid-soluble antioxidants, the

best-characterized groups of compounds in their function of protecting the plant from oxidative stress, plants contain other compounds such as diterpenes displaying high antioxidant properties (Rice-Evans et al., 1997). Rosemary (Rosmarinus officinalis) leaf extracts show a very high antioxidant activity. The main compound responsible for the antioxidant activity is the diterpene, carnosic acid. (Aruoma et al., 1992). Its radical

scavenging activity follows a mechanism analogous to that of other antioxidants such as

-tocopherol and is caused by the presence of two hydroxyl groups in ortho position at

C11 and C12. (Richheimer et al., 1999). Subcellular localization studies show that carnosic

acid protects chloroplasts from oxidative stress in vivo (Munné-Bosch and Alegre, 2001).

31 1.5 The antioxidant potential of extracts and compounds from Pseudopterogorgia

sps.

It has been shown that photosynthetic rates in zooxanthellae can be improved by

the addition of exogenous antioxidants to coral exposed to artificially elevated light and

temperature (Lesser, 1997). Antioxidants improve the rate of O2 evolution by scavenging

free radicals within the dinoflagellate symbiont. Although dinoflagellates can ameliorate

the oxidative burden through the use of endogenous antioxidant enzymes and

photoprotective mechanisms, these systems can be overwhelmed, especially by the synergistic effects of thermal and irradiance stress.

Gorgonians from the Caribbean region are responsible for several diterpenoid carbon skeletal classes including cembranes, asbestinins, briareins, pseudopterosins, pseudopteranes, along with other skeletons that do not belong to any major class. Most of these skeletal classes possess unique functionalities and substitution patterns.

Cembranes represent the largest group of compounds isolated from gorgonians in the

West Indian region (Rodríguez, 1995).

Phenolic diterpenes have been shown to act as potent antioxidant in many

pharmacological studies. Gorgonians of the genus Pseudopterogorgia biosynthesize

many such diterpenes with substantial diversity in both the carbon skeleton and the

location and type of substitutions to the skeleton. The antioxidant potential of these

compounds was assessed using a number of pharmacologically and ecologically relevant

assays. This research attempts to examine whether secondary metabolites from the genus

Pseudopterogorgia act as antioxidants and thus mediate thermal and UV induced

oxidative stress and thereby protect the coral-algal symbiosis. This was accomplished by

32 screening semi-purified extracts as well as pure compounds from these organisms.

Chapter two provides an analysis of the variation in secondary metabolite chemistry of Pseudopterogorgia elisabethae across geographic and depth gradients.

Secondary metabolites vary among species and the biosynthetic pathways producing the compounds are homologous traits which can be used for phylogenetic inference. Indeed some of the most intricate accounts of microevolution have been based on the evolution of secondary metabolites and chemical defense. Efforts to generate chemical fingerprints of samples of the genus Pseudopterogorgia have led to the development of HPLC and

NMR libraries that can be used for the rapid identification of Pseudopterogorgia spp. collected from the northern Bahamas and the Florida Keys. Additionally we have evaluated the variation in secondary metabolites amongst Pseudopterogorgia elisabethae collected from different locations, different depths along the same reef tract and at different times of the year. Pseudopterogorgia elisabethae is currently commercially harvested for the pharmacologically active compounds the pseudopterosins and information about chemical variation across geographic, depth and seasonal gradients may prove useful for future management decisions regarding their harvest.

Chapter three includes results and analysis of assays which screen semi-purified extracts and pure compounds isolated from samples of the genus Pseudopterogorgia for antioxidant potential. First, extracts from seven species of the genus were partitioned into their polar (40% acetone water), intermediately polar (75% acetone-water), and non-polar

(100% acetone) constituents using column chromatography. Then these fractions were then assayed using the ferric reducing ability of plasma (FRAP) assay. The FRAP assay is a measure of "antioxidant power" and offers a putative index of antioxidant, or

33 reducing, potential. In the assay ferric to ferrous ion reduction at low pH causes a colored

ferrous-tripyridyltriazine complex to form. FRAP values are obtained by comparing the

absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions

in known concentration. Based on the results of these initial FRAP assays, it appears that

the intermediately polar fractions of all seven species have antioxidant or reducing

potential, with a significant interspecies variation. The species with the lowest overall

FRAP values was P. hystrix, which according to Bayer (1961) is a deep-water specialist.

Extracts of P. elisabethae where subjected to HPLC and purified compounds were screened in the FRAP assay. These pure compounds had differing antioxidant potentials, and many of those isolated from P elisabethae had activity equal to or greater than

Trolox, a water-soluble vitamin E derivative which is commonly used as a positive control in many antioxidant assays.

In Chapter four the in vitro antioxidant activity of the intermediately polar fractions of seven species of the genus Pseudopterogorgia was tested in a zooxanthellae based assays. This was accomplished using cultured zooxanthellae and the oxidative stress sensitive dye DCFH-DA in a micro-well plate. DCFH-DA diffuses into the cell where cellular esterases cleave the diacetate moiety to form the more polar DCFH, which is trapped within the cell. Oxygen radicals oxidize the intracellular DCFH to the fluorescent DCF. The level of fluorescence measured upon excitation is proportional to the level of oxidation. Antioxidants prevent oxidation of DCFH and reduce the formation of DCF. Extracts (40 µg each dissolved in methanol) were added to wells in triplicate, control wells had either methanol only or nothing added. For all seven species, the intermediately polar fraction significantly reduced fluorescence versus the control cells.

34 Four of the species caused reduction that did not differ significantly from the positive control (Trolox).

Compounds from P. elisabethae previously tested in the FRAP assay were screened using the DCFH-DA zooxanthellae assay. The pseudopterosins caused significant reduction in oxidative state in the cells as evidenced by a significant reduction in DCF fluorescence versus the control, with the pseudopterosin aglycone having the best overall activity (Tukey’s HSD p<0.05).

The ability of secondary metabolites isolated from Pseudopterogorgia elisabethae

to delay photoinhibition in stressed zooxanthellae was also tested. Samples from a clade

B2 Symbiodinium culture from the temperate western Atlantic were subjected to

combined irradiance (light=300µM) and thermal (temperature=34ºC) stress. Pulse-

amplitude modulated (PAM) fluorometry was used to characterize the maximum

quantum efficiency of PSII (FV/FM). Results were compared to control samples to which

no compounds were added and solvent-only controls. Pseudopterosins and especially the

pseudopterosin aglycone seem to reduce the loss of quantum photosynthetic yield over

the nine and a half hour period of the experiment.

In Chapter 5, the in vitro antioxidant activity of secondary metabolites isolated

from samples of the genus Pseudopterogorgia was tested in a mouse sympathetic neuron

cell model. Depriving sympathetic neurons of nerve growth factor (NGF) in vitro leads

to neuronal apoptosis. Reactive oxygen species (ROS) appear a “trigger” in this apoptotic

process- a burst of oxidative stress is detected 4 hr after NGF withdrawal, and superoxide

dismutase (SOD) overexpression inhibits apoptosis. Apoptosis of cells induced by NGF

withdrawal is inhibited by a number of antioxidants and scavengers of ROS.

35 The results presented here are evidence that octocorals may use secondary metabolites to mediate oxidative stress thus reducing the incidence of bleaching. First, extracts from Pseudopterogorgia sps have differing antioxidant potentials as seen using the FRAP assay. Second, diterpenes isolated from P elisabethae exhibit antioxidant activity in FRAP. Third, Pseudopterosins scavenge superoxide radicals generated in mouse neuronal cells deprived of NGF and prevent apoptosis. And finally and most significantly, pseudopterosins (and especially the aglycone) reduce the loss of quantum photosynthetic yield in zooxanthellae exposed to excess heat and light, and reduced significantly the oxidation of the probe DCFH in the zooxanthellae/ micro-plate based assay.

36 CHAPTER 2

Intra-specific chemical variability of Pseudopterogorgia elisabethae.

2.1 Caribbean octocorals

The octocoral fauna of the West Indies is unique in its profusion of gorgonian corals (also known as sea whips, sea fans, or sea plumes). Faunistically, extensions of the West

Indian region reach into the Gulf of Mexico, all the Antilles, the Bahamas, the Florida

Keys, the Bermudas, the Islands of the Caribbean, and south along the northeast coast of

South America to the reefs of Brazil (Bayer, 1961). All over this region the families

Gorgoniidae and Plexauridae flourish as they do nowhere in the world. Gorgonian corals

(order Gorgonacea, phylum Cnidaria) are conspicuous members of most tropical and subtropical marine habitats, being the most abundant octocorals found in the West Indies.

With over 195 species documented from these two major families, gorgonian octocorals represent an estimated 38% of the known fauna (Bayer, 1961). A diverse array of branching octocorals are present on reefs throughout the Atlantic and Indo-Pacific (e.g.,

Fabricius and Alderslade, 2001), and on many reefs in the Eastern Pacific and Western

Atlantic/Caribbean the biomass and diversity of branching octocorals, and in particular

Holaxonians, may be higher than that of scleractinian corals. Octocorals have always been identified as an important component of the reef benthos both in Florida and throughout the Caribbean (Opresko, 1973; Wheaton and Jaap, 1988; Kinzie, 1973; Lasker and Coffroth, 1983; Yoshioka and Yoshioka, 1989; Sanchez, 1998; Sanchez et al., 1999).

37 At many sites, octocorals now are the visually most abundant invertebrate due in large

part to the dramatic loss of scleractinian cover (Porter and Meier, 1992; Hughes, 1994;

Ostrander et al., 2000; Gardner et al., 2003; Cote et al., 2005). Octocorals play additional

roles in benthic ecosystems such as generating sediments (Weinbauer and Velimirov,

1995) and providing habitat for fishes and other invertebrates. Branching octocorals have

also been the subject of intense investigation by natural product chemists, and have

proven to be rich in novel compounds with interesting ecological properties and in some

cases properties of interest to biomedical researchers. Secondary metabolites from

octocorals have been demonstrated to have anti-inflammatory, anti-carcinogenic, and anti-tumor activity (Pawlik et al., 1987; Rodriguez, 1995; Cobar et al., 1997; Mayer et al.,

1998; Shi et al., 2002). While differences in natural product chemistry both between and within taxa have been noted, there has been only limited use of that variability in either the identification or systematics of octocorals. The only attempt to use the chemical data in a systematic context (Gerhart, 1983) identified some of the same ambiguities more recently identified in mtDNA sequence analyses (Sánchez et al., 2003).

2.2 Terpene Biosynthesis

Terpenes, or isoprenoids, are ubiquitous metabolites found in all living organisms.

Sterols, steroidal hormones, and carotenoids are all examples of essential isoprenoids which participate in many important metabolic pathways. Additionally, there are large numbers of terpenes or terpenoids for which physiological roles remain a mystery

(Conolly and Hill, 1992). Terpenes represent the largest family of natural products with over 30,000 members (Buckingham, 1998). They are classified by the number of

38 homologous isoprene units (5 carbon units) in their structure: monoterpenes C10 (2

isoprene units), sesquiterpenes C15 (3 isoprene units), diterpenes C20 (4 isoprene units), sesterterpenes C25 (5 isoprene units), and triterpenes C30 (6 isoprene units).

Terpenoid biosynthesis (Figure 8) involves the head to tail addition of isopentenyl

diphosphate (21) (IPP) units (Ruzicka, 1953). The enzyme isopentenyl isomerase

facilitates the isomerization of IPP to dimethylallyl diphosphate (22) (DMAP). Next, IPP

and DMAP are condensed and the chain elongated by prenyltransferases to create geranyl

diphosphate (23) (GPP- C10). The enzyme-bound geranyl diphosphate undergoes further

condensation with additional IPP units to form larger prenyl diphosphates, namely,

farnesyl diphosphate (24) (FPP- C15) and geranylgeranyl diphosphate (25) (GGPP- C20).

OPP OPP + OPP

22 21

H+ IPP

OPP OPP 23 24 IPP

OPP 25

Figure 8. Biosynthesis of geranyl, farnesyl, and geranylgeranyl diphosphate (23-25) from isopentenyl diphosphate (21) and dimethylallyl diphosphate (22)

39 Cyclization, coupling and/or rearrangement reactions of these prenyl diphosphates forms parent carbon skeletons of monoterpenes, sesquiterpenes, and diterpenes (Singh et al.,

1989; McGarvey and Croteau, 1995; Luthra et al., 1999). These carbon skeletons can then be subjected to structural modification through oxidation, reduction, isomerization, hydration, conjugation, halogenation, and/or other transformations to give rise to a variety of terpenoids (McGarvey and Croteau, 1995).

2.3-Terpenes from Pseudopterogorgia spp.

Caribbean gorgonian octocorals of the genus Pseudopterogorgia are abundant and chemically rich, responsible for the production of several classes of metabolites. Many of the compounds isolated from these marine invertebrates are of great interest because of their structural complexity and their pharmacological potential (Blunt et al., 2005). Two such families of Pseudopterogorgia metabolites are the pseudopterane and the cembrane diterpenoids. Several Caribbean species of Pseudopterogorgia (P. acerosa, P. kallos, and

P. bipinnata) are known to biosynthesize diterpenoids based on the 12-membered carbocyclic pseudopterane skeleton (Tinto et al., 1990; Rodriguez, 1995,). Kallolide A

(14), a major metabolite of P. bipinnata and P. kallos based on the 12-membered carbocyclic pseudopterane skeleton, possesses anti-inflammatory properties comparable to the potency of existing drugs such as indomethacin (Look et al., 1985; Fenical, 1987).

While the pseudopteranes appear to be taxonomically restricted to these

Pseudopterogorgia (Gorgonacea) species and one species of Gersemia (Alcyonacea), the

cembranoids, on the other hand, are often found in many gorgonian and soft coral

species. Diterpenoids of the cembrane class are common metabolites of several West

40 Indian gorgonian genera, especially Eunicea, Plexaura, and Leptogorgia (Rodriguez,

1995). On the other hand, Pseudopterogorgia species that produce cembranoid diterpenes do so in a much more restricted manner. Thus far, cembrane-based diterpenoids have been found in only two of the more than 15 species identified P. acerosa (Tintio et al.,

1990, 1991 and 1995) and P. bipinnata (Fenical, 1987; Wright et al., 1989; Rodriguez and Shi, 1998). Of these, the major producer of cembrane derivatives is

Pseudopterogorgia bipinnata Verrill (family Gorgoniidae), a widely distributed member of this genus. In 1987 Fenical reported that more than 15 cembrane derivatives were isolated from P. bipinnata collected in the Bahamas, (Fenical, 1987). In 1989, Wright et al. reported the isolation and structure elucidation of four cytotoxic furanocembranolides, denoted as bipinnatins A-D (10-13), from a specimen of P. bipinnata also collected in the

Bahamas (Wright et al., 1989). In 1999, Rodriguez and co-workers reported the isolation and structure determination of nine previously unreported pseudopterane and cembrane metabolites from extracts of a Colombian specimen of P. bipinnata. These include bipinnapterolide A structurally related to the known metabolite kallolide A (14) and bipinnatins G-I are highly oxygenated cembranolides which are structurally similar to the known metabolites bipinnatins A-D (10-13). Additionally, Rodriguez et al (1999) isolated from five new related cembranolides bipinnatolides F-J.

Secondary metabolites isolated from gorgonians (sea whips and sea fans) are structurally diverse (Faulkner, 2001) and many exhibit biological activities (Paul, 1992).

Since the polyps of the gorgonians lack physical defenses, the secondary metabolites from these animals have generally been hypothesized to serve as chemical defenses

(Paul, 1992; Pawlik, 1993). During the past decade, laboratory and field experiments

41 have shown that numerous gorgonians possess metabolites that are highly deterrent to

generalist carnivorous fishes. Anti-predatory properties of the crude extracts from 32

species of Caribbean gorgonians were examined in laboratory assays with the generalist

predator Thalassoma bifasciatum and 100% of these crude extracts proved to be deterrent

(O’Neal and Pawlik, 2002). Included in these assays was crude extracts from

Pseudopterogorgia elisabethae, which were among the most potent icthyodeterrent of the extracts tested. Of the gorgonians studied in detail, several metabolites responsible for icthyodeterrent activity have been isolated (O’Neal and Pawlik, 2002).

2.4 Pseudopterosins

Pseudopterosins are an interesting group of diterpene glycosides first discovered by

Fenical and collaborators (Look et al., 1986) from specimens of the gorgonian coral

Pseudopterogorgia elisabethae. So far, 26 pseudopterosins (PsA–PsZ) isolated from specimens collected in the Bahamas (Look et al., 1986), Bermuda (Roussis et al., 1990), the Florida Keys (Ata et al., 2003), and the Columbian Caribbean (Duque et al., 2004) have been reported. The structurally related secopseudopterosins A–D have also been identified in Pseudopterogorgia kallos collected near the Marquesas Keys in Florida

(Look and Fenical, 1987).These pseudopterosins and secopseudopterosins exhibit more potent antiinflammatory and analgesic activities than the common market drug indomethacin (Mayer et al., 1998). It is suggested that the mechanism of action of the pseudopterosins may involve membrane stabilization, different from inhibition of eicosanoid release from inflammatory cells mediated by traditional non-steroidal drugs

(Mayer et al., 1998).

42 Experiments done to elucidate the biosynthetic pathway of pseudopterosins in

Pseudopterogorgia elisabethae present evidence that the pseudopterosin aglycone (26) is the end product of pseudopterosin biosynthesis (Figure 9) and thus may be an active natural product in vivo (Ferns, 2005).

H H H H

OPP OH

GGPP 7- hydroxyerogorgiaene elisabethatriene eragorgiaene

2 20 3 1 H H 9 OH 18 OH H 4 10 8 11 OH 7 12 13 5 O O 6 OH 17 O 14 19 O HO 15 HO

HO HO 16 OH OH amphilectosin A seco-pseudopterosin J 7,8-dihydroxyerogorgiaene

H OH H OH

O OH

O HO

HO OH pseudopterosin Y 26 ps A-D aglycone

Figure 9. Pseudopterosin biosynthesis. Ferns et al., 2005

Perhaps then this represents an example of an activated defense system. Activated defense has been described as a rapid process in which innocuous metabolites that are stored in the tissue are converted to defensive metabolites in response to tissue damage

43 (reviewed in Paul, 1992). The advantage of activated defenses to organisms that possess

them is not one of optimization (because the energy involved in production has already

been expended), but rather the avoidance of autotoxicity. Activated defenses might be

selected over constitutive defenses if the deterrent chemical is physiologically damaging to the producing organism (Baldwin and Callahan, 1993; Wolfe et al., 1997) or if the defensive chemical attracts predators (Carroll and Hoffman, 1980; Giamoustaris and

Mithen, 1995).

The deterrence capacity of pseudopterosins A-D and the pseudopterosin aglycone was tested by incorporating these metabolites into artificial carrageenan-based diets

(Mukherjee, 2005). Paired feeding experiments were conducted on a shallow patch reef

(~3M) at Sweetings Cay, Bahamas. At natural volumetric concentrations (found in the adults colonies collected in Sweetings Cay, Bahamas) pseudopterosins A-D (0.6% dry weight) significantly inhibited feeding relative to controls with high statistical signifigance (Wilcoxon signed rank test 1 tail, p= 0.0062) while the aglycone (0.1% dry

weight) was a significant feeding attractant (p= 0.0027). Based on these results it is hypothesized that the pseudopterosin aglycone has some other ecological role and that its activity as a feeding attractant may explain the low concentration of this metabolite found in undamaged P. elisabethae tissue (Mukherjee, 2005).

The activation of defenses has been widely documented in terrestrial plants (León et al., 2001). Cyanogenic plants contain glycosides composed of a cyanohydrin-type aglycone and a sugar moiety. Cyanogenic glycosides and their hydrolyzing enzymes are stored in different tissue compartments in intact plant tissue. Upon tissue damage, the glycosides are hydrolyzed, and HCN, which is toxic to non-specialist grazers, is released

44 (Vetter, 2000). Additional examples of activated defense in vascular plants include the conversion of phenolic glycosides to phenolics in quaking aspen Populus tremuloides

(Clausen et al., 1989) and balsam poplar Populus balsamifera (Reichardt et al., 1990), and the hydrolysis of glucosinolates to form thiocyanates, isothiocyanates, or isonitriles in the Cruciferae and related plants (Van Etten and Tookey, 1979; Chew, 1988). There are examples of activated defense in marine organism as well. Van Alstyne and Paul

(1992) found that most species of Halimeda on Guam immediately convert the less- deterrent secondary metabolite halimedatetraacetate to the more potent feeding deterrent halimedatrial upon injury simulating fishes feeding on Halimeda plants. Extracts from injured plants contained higher amounts of halimedatrial and were more deterrent toward herbivorous fishes than extracts from control plants (Van Alstyne and Paul, 1992).

Cetrulo and Hay (2000) investigated the frequency of activated chemical defenses in seaweeds using urchin and fish feeding assays of chemical extracts from 42 species of seaweeds that were damaged immediately before extraction in organic solvents (the potentially activated extract) versus extracts from undamaged tissue (the non-activated extract). Seven species exhibited changes in palatability consistent with activated defenses while 4 species became more, rather than less, palatable if they were damaged

30 s before extraction (Cetrulo and Hay, 2000).

Identifying the true biosynthetic origin of secondary metabolites isolated from marine organisms is confounded by the fact that most marine organisms live in tight symbiotic association with many marine microbes. Over the past decade, there has been a growing awareness that compounds whose biosynthesis had been attributed to marine invertebrates may actually be produced by a symbiont (Vacelet and Donadey, 1977;

45 Anthoni et al., 1990; Kobayashi and Ishibashi, 1993). From both ecological and

biomedical viewpoints, it is important to know whether the metabolites isolated from

marine organisms are produced by symbiotic microorganisms (Faulkner, 1994). Most

gorgonian species form obligate associations with dinoflagellates of the genus

Symbiodinium in order to survive in the nutrient-poor tropical waters. Nutrition, in the

form of translocated photosynthates, is the primary contribution of the dinoflagellate to

the host. The dinoflagellate receives inorganic nutrients as well as protection from the

intense predation of marine environment in return (Whitehead and Douglas, 2003). For

corals which are endowed with symbiotic zooxanthellae, there is debate whether the coral itself, the zooxanthellae, or another microbial associate is the source of the secondary metabolites. Historically, terpenes have been viewed as plant metabolites; thus zooxanthellae were assumed to be the source of these compounds.

The diterpene crassin acetate was purified from the extract of zooxanthellae isolated from the gorgonian crassa (Ciereszko, 1962), and diterpenes have been purified from the algal symbiont of P. elisabethae and P. bipinnata (Mydlarz et al, 2003; Boehnlein et al, 2005). Conversely, isolation of terpenoids from

azooxanthellate Lophogorgia alba (Bandurraga et al., 1982) and azooxanthellate

Lobophytum compactum (e.g. Michalek-Wagner et al, 2001) gives credence to terpene

biosynthesis being under animal control. In the bryozoan Bugula neritina, biosynthesis of

the bioactive bryostatins has been localized to bacterial symbionts (Davidson and

Haygood, 1999). Research on the sponge Dysidea herbacea shows that a unique group of

polychlorinated compounds isolated from the whole sponge tissue is limited to the

46 cyanobacterial filaments, whereas the accompanying sesquiterpenoids are found only in

the sponge cells (Unson and Faulkner, 1993).

The larvae of the gorgonians Pseudopterogorgia elisabethae and P. bipinnata

lack algal symbionts. Examining these for the presence of diterpenes, and assessing their

biosynthetic capacity can therefore provide additional information about the true source

of these ecologically and pharmacologically important secondary metabolites.

Hildebrand et al (2004) suggest the use of the following criteria when attempting to

determine the source organism of a metabolite: 1) correlate presence of the symbiont with

a function for the host, 2) remove the symbiont and show loss of function, 3) reintroduce

the symbiont and show that function is regained, and 4) isolate the symbiont again. One

difficulty lies in obtaining symbiont-free hosts, as many marine organisms are not viable

without their symbiont. Also, isolating symbionts for reintroduction can be complicated, as obligate symbionts may not be culturable.

Since most organisms have many microbes associated with them, it is important to

determine which of these are persistently associated with the host (Hildebrand et al

2004). When there are variations in natural products within a host species or related

group of species, correlation of the presence of a particular microbe with a particular

compound provides support for the microbial involvement in synthesis of that compound.

If animals lacking a particular microbe also lack the metabolite, this suggests that the

microbe might be a good candidate for further study. In some cases, symbionts can be

reduced or eliminated by treatment with antibiotics or by other methods. Correlation of

symbiont reduction or elimination with reduction or elimination of the metabolite can

provide useful information.

47 Reproductive tissues in gametes and larvae are always important to examine for

the presence of symbionts. Although symbionts can be recruited from the environment, in

many cases, the host has evolved mechanisms to ensure vertical transmission. Microbes

persistently associated with eggs and larvae are likely to have important roles in the life

of the host, one of which could be synthesis of bioactive metabolites (Lopanik et al,

2004).

Biosynthetic experiments can be performed in different ways. One of these is

feeding with an isotopically labelled precursor in order to prove whether this is

incorporated into the final product or not. A radiolabeled (e.g. 3H or 14C) putative

precursors can be used in in vivo or in vitro assays to monitor the transformation of a known precursor to the secondary metabolite of interest. In the case of terpene

3 biosynthesis, the radiolabeled precursor [ H-C1]-GGPP can be used. Radiolabeled

isotopes are extremely sensitive and easily detected by scintillation counting. This

sensitivity is useful when the enzymatic turnover rate from precursor to secondary metabolite is low. When a radiolabeled precursor is being employed radiochemical purity for the isolated secondary metabolite must be assessed after isolation of the target compound. This can be done by derivatization of the compound followed by purification of the derivative and monitoring of specific activity. Finally, in order to conclude that a label was incorporated into a metabolite, radioactivity at least twice background (an inactive sample) should be obtained for the metabolite of interest.

Chemical analysis of the azooxanthellate larvae from the sea whips

Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata revealed higher concentrations of diterpenes than are present in the intact holobiont.

48 Table 1. Comparison of pseudopterosin A-D and kallolide A Acetate content from P. elisabethae and P. bipinnate expressed as % organic extract. Larvae Polyps Zooxanthellae Holobiont Pseudopterosins 30.39 ± 1.48 1 N/A 11 2 5 2 A-D Kallolide A 21.51 ± 0.97 1 18.75 ± 2.11 1 7.3 2 4.27 2 acetate 1 Mukherjee, 2005, Average of n = 3 experiments ± st. dev. 2 Mydlarz et al., 2003 3 Boehnlien et al., 2005

Biosynthetic studies of the larvae with 3H-labeled geranylgeranyl diphosphate

indicated that they are capable of diterpene biosynthesis. Previous studies had suggested

that pseudopterosin and kallolide biosynthesis proceeds in the absence of P. elisabethae

(Mydlarz et al., 2003) and P.bipinnata (Boehnlien et al., 2005) cells. In these studies,

zooxanthellae were separated using a gravimetric gradient of Percoll. To further investigate whether this is indeed the case, DNA from freshly isolated zooxanthellae, and

DNA from the larvae (from each of these two host organisms) was subjected to PCR reactions with zooxanthellae specific primers (Santos et al., 2001) and

Pseudopterogorgia specific primers (Gutierrez-Rodriguez et al., 2004). The reaction resulted in the amplification of a fragment of DNA corresponding to the presence of both zooxanthellae and coral DNA from the isolated zooxanthellae. The larvae DNA from both species clearly lacked algal DNA, as no amplicon was seen corresponding to the expected product of the zooxanthellae primers (Figure 10).

It is quite possible that results of previous studies, in which biosynthesis appeared to occur in the absence of coral cells, may have been confounded by the presence of coral cells in what was thought to be purified algal symbionts. Coral DNA was found in

49 zooxanthellae isolates regardless of the number of times the purification procedure was repeated, up to three consecutive times. In an attempt to further localize the biosynthesis,

larvae zoox P.e. larvae zoox P.e. N.C. DNA DNA DNA DNA DNA DNA

Figure 10. An example of an agarose gel showing amplification products from larvae, isolated zooxanthellae, and holobiont DNA using zooxanthellae and Pseudopterogorgia specific primers.

larvae from both species were treated over 48 hours with a combination of antimycotic

and antibiotic agents, following a previously published procedure used successfully in

with the larvae of the bryazoan Bugula neritina (Lopanik et al., 2004). Briefly, For each

species, n=3 containers of 100 ml MFSW with ~100 larvae were treated with antibiotic/antimycotic twice over 48 hours with final concentrations as follows: 0.1 mg/ml kanamycin (a water-soluble, broad-spectrum antibiotic), 0.1 mg/ml streptomycin

(active against both gram-positive and gram-negative bacteria, including species resistant to other antibiotics), 100 units/ml penicillin, 0.25 g/ml amphotericin B. At the end of the

48 hours the samples were incubated for 24 hrs with the tritiated diterpene precurser and then extracted and analysed as previously described. Treatment of the larvae from both

50 species with antimicrobial agents did not reduce their capacity to synthesize these

bioactive compounds (Table 2). Radiochemical purity of the pseudopterosin Y and

kallolide A acetate was verified via derivitization reactions as previously described

(Mydlarz et al., 2003 for pseudopterosins; Boehnlein et al., 2005 for kallolide A acetate;

Mukherjee, 2005). In each case significant change in structure and physical properties

between the parent compound and the derivative resulted in a significant retention time

difference of over five minutes on both normal and reversed phase HPLC, ensuring that

the associated radioactivity was due to the compound of interest and not the co-elution of

a highly radioactive impurity. Structures of parent compounds and derivatives were

confirmed by 1H- NMR.

Table 2. Comparison specific activities of pseudopterosin and Kallolide A acetate from larvae treated with antibiotics and antimycotics and those untreated. Average of n = 3 experiments ± st. dev.

Specific Activity (DPM/mmol) P. elisabethae larvae P. elisabethae larvae Ab/Am treated

pseudopterosin Y 2.4 ± 0.58 E7 2.8 ± 0.23 E7

Specific Activity (DPM/mmol) P. bipinnata larvae P. bipinnata larvae Ab/Am treated

Kallolide A Acetate 3.6 ± 0.89 E6 5.6 ± 1.22 E6

Based on these results, it seems likely that the terpenoid biosynthesis occurs

within the coral cells. Further studies with these early ontegenetic tissues should include thorough examination of antibiotic and antimycotic treated larvae to confirm that they are free of microbial associates.

51 2.5 Variability of pseudopterosin content across a geographic gradient

Patterns of intraspecific variation in composition and concentration of secondary metabolites among geographic regions or habitats have been well documented for terrestrial plants (Gershenzon and Croteau, 1991). A few studies have shown variability in secondary metabolite content within or between individuals of a given species (e.g.,

Meyer and Paul, 1995; Maida et al., 1993; Harvell et al., 1993; Becerro et al., 1998;

Amade and Lemee, 1998). Some of these reports have correlated the variation to spatial variables, such as depth or geography. For example, Maida et al. (1993) found significant differences in the major metabolites, flexibilide and sinulariolide, of the soft coral

Sinularia flexibilis among sites at Lizard Island (Great Barrier Reef, Australia). In the

Caribbean, Harvell et al. (1993) detected variation in composition and concentration of the major metabolites of the gorgonian Briareum asbestinum among depths and geographical sites. In a study done by Benayahu and co-workers (2000) intraspecific variation in composition of secondary metabolites of Parerythropodium fulvum fulvum, a common encrusting alcyonacean soft coral on reefs as well as in concentrations of its major metabolites were correlated with different color morphs and varying depths.

The great degree of chemical variation among different specimens of P. elisabethae from various sites in the Caribbean region has been acknowledged by several authors. Different kinds of the closely related pseudopterosins have been isolated from

Bahamian, Bermudan and Columbian specimens of P. elisabethae (Look et al., 1986;

Roussis et al., 1990; Puyana et al., 2004). So far, it is not known if chemical variability and pseudopterosin production are genetically or environmentally determined.

52 Fragments of P. elisabethae colonies were collected by SCUBA at various sites and depth ranges around the islands of the Bahamas and Key Largo, Florida (for GPS coordinates see Table A1). Sample collection never involved removing whole colonies, only a terminal fragment of each individual colony was cut from the main gorgonian axis with sharp scissors. Gorgonian fragments were air-dried, labeled, and stored in the freezer. Samples were kept frozen until extraction. Colony fragments used for chemical analyses were collected at 10 sites around the Bahamas and 1 site off Key Largo between

May of 2000 and September of 2007 (Figure 11). We collected on average 10 replicates per location. The morphology of P. elisabethae colonies can be highly variable.

Therefore, to avoid confusion with other species, we corroborated the identity of each colony by comparison of the gross colony architecture and by morphology and dimensions of the three most common sclerite types (i.e., spindles, anthocodial rods, and scaphoids). To obtain sclerites, we clipped off a small fragment of the colony and digested it in household bleach. Once the organic tissue was digested, sclerites were repeatedly washed with distilled water and ethanol, and dried. Crude sclerite preparations were mounted on glass slides and analyzed under a microscope. For each colony, we measured 30 anthocodial rods, 30 spindles, and 30 scaphoids. For species designations, we followed Bayer (1961). Based on morphological analysis, all colonies collected were correctly identified in the field as P. elisabethae.

53

Figure 11. Collection sites of Pseudopterogorgia elisabethae in the Bahamas and Florida. Detail shows the sites clustered along the Little Bahamas Bank.

Each dried colony was cut in small pieces and weighed. 1 g of gorgonian tissue was extracted with methanol. Crude extracts were analyzed by thin layer chromatography

(TLC) on silica plates with 40:60 hexanes:ethyl acetate as the developing solvent. Plates were visualized under UV254nm light followed by charring with 10% H2SO4 in ethanol.

Characteristic TLC profiles were obtained for each location. The overall morphology of

P. elisabethae can be variable, and chemical differences were not correlated to specific

54 morphs. We confirmed species identity of each colony by morphological and sclerite

analysis and found no significant differences in sclerite dimensions among different

colonies. Therefore, we ruled out the possibility that we were dealing with different

species.

A portion of each crude extract was redissolved in methanol for additional

purification. Methanol extracts of colony tissue are fractionated on the polymeric

chromatographic support HP20. Dilation HP20 Poly(styrene-divinylbenzene) resin is

used to create discrete fractions that can be examined using HPLC and NMR. The

extract is pre-fractionated in three fractions, a polar, a medium-polar and a non-polar

fraction. Most biologically active compounds need to be able to travel throughout a

target organism’s body in order to reach the intended sites of action. They therefore need

to be soluble in both hydrophilic and hydrophobic environments (Zubay, 1998).

Consequently a majority of biologically active compounds are neither extremely polar

nor non-polar in character (possess intermediate polarity). We have found that by using

the polymeric chromatographic resin Diaion HP-20 instead of traditional silica based

supports (Silica gel, C18 etc.) medium-polar biologically active secondary metabolites

such as terpenoids can be efficiently separated from water-soluble carbohydrates, salts,

and non-polar fats and steroids that are found in high concentrations

We then employed HPLC analysis of the medium-polar fraction to obtain a characteristic chemical profile for each colony. HPLC profiles allowed us to confirm chemical differences among colonies of P. elisabethae collected from different sites.

Chromatograms indicated five chemotypes (Figures 12, 13 and14), each with a distinct pattern of peaks with varying retention times. For simplicity we will refer to the

55 chemotypes as follows, Burrows/San Salvador (BSS), Burrows/Little Bahama Bank

Hybrid (BLH), Little Bahama Bank (LBB), Exuma Sound/Bimini (ESB) and Key Largo

(KLP). Analysis of replicate samples from within each site showed consistent results,

with the exception of the Burrows site, where we have identified two chemotypes.

Although quantities of individual metabolites varied and some minor metabolites were absent in some cases, the general secondary metabolite profiles were consistent for each

chemotype (Table 3). Quantification of pseudopterosins in crude extracts was performed

by HPLC with UV detection, using a calibration curve generated with pure

pseudopterosin A as a standard. All known pseudopterosins contain a similar

chromophore facilitating their quantification by chromatographic methods using one

standard.

The Bahamas Archipelago consists of vast carbonate banks (average depth ~ 3 m)

with islands, deep channels, and deep-water basins. The collections sites were as follows.

Cross Harbor, Long Rock, Gorda Cay, Sandy Point and AB102 are clustered along a ~ 20

km stretch of the Little Bahamas Bank off the coast of Abaco Island at the northern end

of the chain of Bahamian islands. Burrows is located at the northern end of Little Bahama

Bank, separated from the other sites by ~20 km. Little San Salvador, Cat Island and Hog

Cay are in Exuma Sound. Exuma Sound and Little Bahamas Bank are separated by the

Northeast Providence Channel. The Northeast Providence Channel ~4000 m depth

extends south to form the Tongue of the Ocean. The channel is about 40 km wide. The

distance between the northern end of Cat Island and San Salvador is about 135 km. The

open waters of the western North Atlantic Ocean lay in between. San Salvador Island lies

56

A B

BSS 57

C L

BHY PKL Figure 12. HPLC chromatograms of chemotypes of P. elisabethae-BSS chemotype: A = San Salvador, B = Burrows; BHY chemotype C= Burrows; PKL chemotype, L= Pickles Reef.

D F

E 58 G

ESB

Figure 13. HPLC chromatograms of chemotypes of P. elisabethae- ESB chemotype: D= Cat Island, E= Hog Cay, F= Little San Salvador, G= Bimini.

H I

59 JK

LBB Figure 14. HPLC chromatograms of chemotypes of P. elisabethae LBB chemotype, H= Gorda Rock, I= Long Rock, J = AB102, K= Sandy Point on a small, isolated bank at the eastern edge of the Bahamas. San Salvador represents both the eastern most and southern most site in our survey. Pickles Reef, off-shore of Key

Largo, is separated from Bimini by the Straits of Florida channel, with a depth of about

800 m and a width of ~85 km. Bimini is on the northwest corner of Great Bahama Bank, and is separated from the sites along Little Bahama Bank by the Northwest Providence

Channel.

The distribution of the five chemotypes across this geographic region does not lend it’s self to an obvious interpretation. The sites clustered along the Little Bahama

Bank display the same chemical profile (LBB), while Burrows, just ~20km north of the northern most site of this group has two chemotypes. BSS is distinctly different, and the

BLH is essentially a hybrid of the BSS and LBB. It has previously been reported

(Mukherjee, 2005; Kohl and Kerr, 2003) that P. elisabethae from Sweetings Cay,

Bahamas are of the BSS chemotype. Sweetings Cay is ~ 10 km further north along the

Little Bahama Bank from Burrows. San Salvador, the southern most site in our survey, is over 300 km from Burrows, yet colonies from these sites share the BSS chemotype.

Colonies from the geographically disconnected sites in Exuma Sound and Bimini share the ESB chemotype. Additionally, P elisabethae from Pickles Reef off Key Largo,

Florida produces the aglycone from which the pseudopterosins of the ESB chemotype are derived.

One of the central paradigms in the study of marine life history strategies is the relationship between the number of eggs/larvae produced, their size and mode of development (Thorson, 1950). A corollary to that relationship is that large lecithotrophic and brooded larvae do not disperse as widely as planktotrophs. That in turn, suggests

60 reduced connectivity and greater susceptibility to local extinctions among brooding

species. Hughes et al. (1999) found differences in the patterns of spatial variance of

brooding and broadcast spawning species, and Underwood and co-workers (2006) report

that brooding by the coral Seriatopora hystrix has led to limited connectivity among

Western Australia reefs. Sexual reproduction in P. elisabethae and P. bipinnata occurs

via surface brooding. Fertilized (and possibly unfertilized) eggs are maintained in a

mucus layer on the surface of the maternal colonies and develop there into competent

planulae (Gutierrez-Rodriguez and Lasker, 2004). We might predict that P. elisabethae

would have limited dispersal extending to zoogeographic barriers.

Figure 15. Map of the hydrography of the collections region.

The purified compounds were identified by UV, CD, MS, 1H, and 13C NMR spectroscopy (Appendix), and by comparison of our spectral data with those previously reported (Look et al., 1986; Look and Fenical, 1987; Roussis et al., 1990; Puyana et al.,

2004). This research has yielded six previously undescribed pseudopterosin. All were

61 isolated from the samples collected at Cross Harbor, Long Rock, Gorda Cay, Sandy Point and AB102 (Table 1). Two novel variants of the previously reported pseudopterosin K were recovered. These differ from pseudopterosin K (35) only in the presence and location of an acetate group on the sugar moiety of the molecule. While the fucose of pseudopterosin K is not acetylated, the new ps K variants have an acetate group at carbon two (ps K 2’ acetate; 36) and carbon four (ps K 4’ acetate 37) of the sugar ring.

Additionally, a series of novel pseudopterosins, the iso-pseudopterosins A-D were also isolated from samples collected at these sites. The iso-ps A-D series (30-33) are stereochemically identical to the previously described ps A-D (17-20) series (Look et al.,

1986), but the glycoside linkage is at C-10 of the diterpene skeleton, not C-9 as on the ps

A-D series (NMR data table A9).

Figure 16. Structures of pseudopterosins G-K, iso A-D and ps G-J aglycone.

62

63 Chemical diversity in soft corals may have an adaptive value in mediating

ecological interactions in reef environments (Sammarco and Coll, 1988). Several studies

have demonstrated that interactions such as predation (Pawlik et al., 1987; Puglisi et al.,

2000; Thornton and Kerr, 2002), competitive and allelopathic interactions (De Nys et al.,

1991; Maida et al., 1993, 1995; Leone et al., 1995), and reproductive state (Coll et al.,

1989, 1995) may influence the type and quantities of specific compounds produced by

soft corals. When trying to understand chemical variability, the issue is complicated by the influence of ecological variables such as habitat characteristics and depth (Harvell et al., 1993; Roussis et al., 2000; Thornton and Kerr, 2002). At this point, we do not know the causes for the extreme chemical variability of P. elisabethae in the areas we surveyed.

Thornton and Kerr (2002) found that predation by the ovulid gastropod Cyphoma

gibbossum, or decreased levels of UV radiation had a significant effect on the

concentration of pseudopterosins while simulated wounding or predation by the butterfly

fish Chaetodon capistratus did not. Interestingly, the greatest increase of pseudopterosin

concentration occurred in response to decreased UV radiation. The authors argue that

pseudopterosin biosynthesis may involve, directly or indirectly, a photosynthetic

organism (probably zooxanthellae). A great diversity of zooxanthellae has been

recognized in hermatypic corals (Rowan and Knowlton, 1995; Toller et al., 2001). This

diversity is closely related to environmental variables and can change in response to

environmental stress. P. elisabethae harbors zooxanthellae (Rowan, 1988), and one

would expect to find different symbionts on a wide geographical range (i.e., the

Caribbean). A study done by Santos and co-workers (2003) used microsatellites to

characterize the Symbiodinium sp. clade B populations hosted by the gorgonian

64 Pseudopterogorgia elisabethae in the Bahamas. Many of the collection sites for their study are the same as those presented here. Twenty-three unique, two-locus genotypes were identified in association with these P. elisabethae colonies. Most colonies hosted only a single Symbiodinium sp. clade B genotype; however, in some instances (n=25), two genotypes were harbored simultaneously. For 10 of the 12 populations, 66–100% of the P. elisabethae colonies hosted the same symbiont genotype. Added to this, in 9 of the

12 populations, a Symbiodinium sp. clade B genotype was either unique to a population or found infrequently in other populations (Santos et al., 2003). These distinct symbiont genotypes do not correlate well with the chemotype data we have generated, and it seems highly unlikely that chemical variation is a result of symbiont variation.

Genetic analysis of the adult populations of P. elisabethae (Lasker, unpublished data) identified small but significant θ and ρ values (estimators of Fst and Rst) among the

Little Bahama Bank (LBB) populations. According to this analysis, the data best fit a model of 4 populations, one being Little San Salvador, one Exuma Sound and Florida, and the other two distributed across the New Providence Channel from Bimini on the east to Southampton reef to the west. The New Providence Channel collections were made up of individuals whose genotypes are admixtures of 2 populations.

Interestingly, Lasker found that Hog Cay (HC) and Pickles Reef, Florida (FL) form a homogeneous population, and we found that the chemistry from these two geographically distant sites is also related, as the pseudopterosins from Exuma Sound and the pseudopterosin aglycone from Pickles reef are identical. Lasker concludes that that while the genetic analyses have identified clear structure between the New Providence

Channel, San Salvador, Exuma Sound and Florida, the 5 loci currently in use do not have

65 the power to clearly assess differentiation among the Little Bahama Bank populations

(Lasker, personal communication).

The intra-species chemical variation we have found in P elisabethae may likely

represent phenotypic plasticity. Phenotypic plasticity describes the capacity of a genotype

to exhibit a range of phenotypes in response to variation in the environment. Single

genotypes can change their chemistry, physiology, development, morphology, or

behavior or in response to environmental cues (Agrawal, 2001). Environmental variation

encompasses both abiotic and biotic components of the environment, including

interactions among organisms. The strength and outcome of many ecological interactions,

ranging from antagonism to mutualism, are mediated through the phenotypically plastic

responses of one or more players in the interaction (Fordyce, 2006). The conditions favouring phenotypic plasticity are certainly found on coral reefs, that is, sessile corals experience disruptive selection both in space and time (Bradshaw, 1965; Potts, 1984).

The dispersal of larvae, with little motility and limited capacity for substrate selection, into the heterogeneous reef environment almost eliminates any chance of predicating

what habitat offspring will settle in. Plasticity is important in the maintenance of natural

populations, enhancing the survival and reproductive success by contributing to their

ability to cope with environmental changes and to potentially adapt to new niches.

Adaptive plasticity describes phenotypic responses to the environment that results in

increased fitness for an organism, such as inducible defenses of plants in response to

herbivore attack (Karban and Agrawal, 2002). Some phenotypic plasticity is clearly

adaptive however, this is difficult to demonstrate empirically (Newman, 1992).

66 Reciprocal transplant experiments of Todd and co-workers (2004) clearly demonstrated phenotypic plasticity in the morphology of two species of Indo-Pacific corals and there relation to environmental factors such as depth and sedimentation rates.

If larval settlement in benthic habitats is random, the developing organism has the ability to generate the phenotype that conveys the greatest fitness for the local conditions. The phenotype is then acquired through development under the current environment and can be changed in the next generation, if conditions are modified (Todd et al., 2004).

In summary, the present study reveals a qualitative variation in the secondary metabolite content of P. elisabethae between collection sites. Although the potential adaptive nature of such variation is still unknown, it could well be in response to some yet undescribed and localized selection pressure. Higher resolution genetic data may lead to a closer correlation of chemotypes and population differences. Pseudopterogorgia elisabethae is currently commercially harvested for the pharmacologically active compounds the pseudopterosins, and information about chemical variation across geographic, depth and seasonal gradients may prove useful for future management decisions regarding their harvest. Future research in this area should examine between site variation in environmental parameters such as predation pressure, pathogen abundance, UV irradiance and temperature. Furthermore, one should take intraspecific variation into account when presenting a chemical fingerprint of a given species.

67 CHAPTER 3

Antioxidant potential of extracts and pure compounds from Pseudopterogorgia sps.

3.1 Antioxidants and marine organisms

One potential ecological role for the diverse and novel secondary compounds produced by gorgonians may be defense against oxidative stress. In a study done during the height of the 1998 mass bleaching event on the Great Barrier Reef, the octocoral families Briareidae, Clavularidae, Gorgonidae, and Nephtheidae exhibited low bleaching susceptibilities (Goulet et el., 2008). There was no indication that different

Symbiodinium populations explained the occurrence of bleached or unbleached tissues.

The authors of this study concluded that differential tolerance to stress among cnidarian species, irrespective of the Symbiodinium they host, may explain differences in bleaching.

In the Caribbean, octocorals are the most conspicuous reef macrofauna, with up to

40 species co-occurring in a single area (Sánchez et al., 2003). A striking difference between Caribbean octocorals and other zooxanthellate cnidarians is their seemingly greater resistance of bleaching. While there have been numerous reports of scleractinian coral bleaching throughout the world’s tropical oceans, there have been far fewer observations of bleaching among octocorals (Lasker, 2003).

It has been shown that photosynthetic rates in zooxanthellae can be improved by the addition of exogenous antioxidants to coral exposed to artificially elevated light and temperature (Lesser, 1997). Antioxidants improve the rate of O2 evolution by scavenging

free radicals within the dinoflagellate symbiont. Dinoflagellates can ameliorate this

68 oxidative burden through the use of endogenous antioxidant enzymes and photoprotective mechanisms but these systems can be overwhelmed, especially by the synergistic effects of thermal and irradiance stress.

Seaweeds or their extracts have been studied as potential natural antioxidants during the last decade (Anggadiredja et al., 1997; Duval et al., 2000; Lim et al., 2002;

Matsukawa et al., 1997; Ruberto et al.,, 2001; Ruperez et al., 2002; Tutour et al., 1998;

Xue et al., 2001; Yan et al.,1998). Although most photosynthesizing plants including seaweeds are exposed to a combination of light and high oxygen concentrations, which lead to the formation of free radicals and other strong oxidizing agents, they seldom suffer from any serious photodynamic damage in vivo. Relatively few attempts have been made to explore this vast resource of structurally unique chemistry for new antioxidant prototypes. Marine antioxidant research has largely focused on the antioxidant effects of crude extracts (Guzman et al., 2001). The previously characterized marine antioxidant substances are mainly chemicals that are structurally related to plant-derived antioxidants

(Nakagawa et al., 1998; Son et al., 2002). These marine antioxidants include pigments such as chlorophylls (Yamamato et al., 1992; Wantanabe et al., 1993) and carotenoids

(Schubert et al., 2006) and tocopherol derivatives such as vitamin E and related

isoprenoids (Yamamato et al., 2001). Certain phenolic substances produced by marine

algae (Fujimoto and Kaneda, 1984) have been shown to have antioxidant activity. UV-

absorbing mycosporine- like amino acids found in marine microalgae and invertebrates

have been shown to have antioxidant activity (reviewed in Klisch and Häder, 2008).

Griffin and Bhagooli (2004) showed that the ferric reducing antioxidant power

(FRAP) assay could be a useful tool in determining antioxidant potential in corals. Their

69 results clearly demonstrated that when corals are exposed to thermal stress, an increase in

enzymatic antioxidant concentrations can be measured using the FRAP assay. Other

research on coral has shown an increase in individual enzymatic antioxidant

concentrations (lipid peroxidase, MnSOD and Cu/ZnSOD) in corals after being subject to

thermal stress (Dykens and Shick, 1982; Lesser, 1996; Downs et al., 2000 and Downs et al., 2002). The FRAP assay can provide information on total antioxidant potential in a relatively short time without having to run more lengthy tests for each individual

antioxidant. It is demonstrated here how this assay could be a simple, reproducible and inexpensive tool for marine biologists involved in ecotoxicological studies.

3.2 Ferric reducing antioxidant power (FRAP) assay.

Benzie and Strain (1996) considered the antioxidant as any species that reduces

the oxidizing species that would otherwise damage the substrates. And the authors further

treat the "total antioxidant power" as the "total reducing power". The antioxidant activity

is then interpreted as the reducing capability. Total antioxidant activity is measured by

ferric reducing antioxidant power (FRAP) assay. FRAP uses antioxidants as reductants in

a redox-linked colorimetric method, employing an easily reduced oxidant system present

in stoichiometric excess. At low pH, reduction of ferric tripyridyl triazine (Fe III TPTZ)

complex to ferrous form which has an intense blue color can be monitored by measiring

the change in absorbance at 593nm. This reaction is non specific, in that any half reaction

has lower redox potential, under reaction conditons, than that of ferric ferrous half

reaction, will drive the ferrous (Fe III to Fe II ) ion formation. The change in absorbance

is therefore directly related to the combined or total reducing power of the electron

70 donating antioxidants present in the reaction mixture. The relevant chemical reaction of

the FRAP method involves a single electron reaction between Fe (TPTZ)2 (III) and a

single electron donor ArOH (Prior et al., 2005)

+· Fe(TPTZ)2(III) + ArOH → Fe(TPTZ) 2(II) + ArOH

The FRAP assay can be performed on a wide range of complex biological fluids,

including plasma, serum, saliva, tears, urine, cerebrospinal fluid, exudates, transudates,

and aqueous and ethanolic extracts of drugs, foods, and plants, as well as on simple and

heterogeneous solutions of pure antioxidants (Benzie and Strain, 1996).

A major advantage of the FRAP method is its ability to quantify the amounts of

total antioxidants or reductants in extracts. The FRAP assay is also the only one to directly measure antioxidants or reductants in a sample. Other commonly employed assays are more indirect because they measure the inhibition of reactive species (free radicals) generated in the reaction mixture. These results are strongly dependant on the type of reactive species used (Guo et al., 2003). The FRAP assay, in contrast, uses antioxidants as reductants in a redox-linked colorimetric reaction. Moreover, the other assays (but not FRAP) use a lag phase type of measurement. In previous experiments this has been difficult to standardize and has generated different results between different laboratories. In the FRAP assay, pretreatment is not required, stoichiometric factors are constant and linearity is maintained over a wide range (Benzie and Stezo, 1999).

In order to streamline the analysis of large collections we have developed a small- scale standardized solid phase extraction (SPE) and HPLC procedure to allow the rapid fractionation of multiple samples and generate HPLC data that can used to prioritize

71 samples for scale-up isolation. The crude fractionation of the extract is achieved using a

solid phase extraction (SPE) 12-port vacuum manifold to allow fractionation of multiple

samples using HP-20. The extract is fractionated in three fractions, a polar, a medium-

polar and a non-polar fraction. The objective of this step is to remove a majority of the very polar salts and carbohydrates and very non-polar fats and steroids in the crude extract. These pre-fractionated “enriched” materials can then be examined using NMR for the presence of novel compounds or screened for biological activity.

Initial separation of methanolic extracts of Pseudopterogorgia sps material were partitioned into three discrete fractions of decreasing polarity using the reverse-phase HP-

20 chromatographic method described above. We have taken these three discrete fractions (40%, 75% and 100% Acetone:H2O) and examined them for antioxidant

potential using the FRAP assay. Samples of each fraction (100 µg) were loaded in

triplicate onto 96-well microtitre plates with FRAP reagent. Activity was determined per

μg of sample using a linear calibration curve generated from FeSO4·7H2O standards of

known concentrations. Aqueous solutions of known Fe(II) concentration (FeSO4 ·7H20) were used for calibration of the FRAP assay. Reaction of Fe(II) represents a one electron exchange reaction and is taken as unity, i.e., the blank corrected signal given by 100 µM solution of Fe(II) is equivalent to a FRAP value of 100 µM. Typical Fe(II) standard concentrations used in these experiments are in the range of 50-1000 µM (Figure 17).

Results from these initial experiments correlate with our assumption that the intermediately polar fractions (75%) contain the majority of the biologically active compounds (Figure 18).

72 FeSO4 Standard Curve 1.4 y = 0.0012x - 0.0387 R2 = 0.9972 1.2

1

0.8

0.6 Absorbance

0.4

0.2

0 0 200 400 600 800 1000 1200

M FeSO4

Figure 17. An example of a standard curve generated using concentrations of FeSO4

Antioxidant Potential of Pseudopterogorgia sps extracts

20

18

16

14

12

g extract 40 

/ 10 75 4 100 8

M FeSO 6 

4

2

0

) ) ) ta is a ) sa s H) id sp L o gida llos n g (KL (CH) nna er ri a e (C ri e i ae (B hystrix hystrix c k ill ta s (KL) h ipinnata a ae (LR u ae a o ha bip b h q h n t n t n rigida (K e a e i kall sabethae b(B) l b ip li b a sabethaeb (KL) e lisa elisabet lis li alpha tocopherol e elisabet e e

Figure 18. FRAP analysis of polar (40%), medium-polar (75%) and non-polar (100%) fractions of the crude extract of seven species of the genus Pseudopterogorgia.

Vitamin E is a generic name for all naturally occurring tocopherol and tocotrienol derivatives, among which α-tocopherol exhibits the highest antioxidant activity (Fryer,

73 1993). It is well recognized that vitamin E (α-, β-, γ-, δ-tocopherol) is an outstandingly

effective antioxidant and can scavenge active free radicals (Mukai et al., 1988). The study

of Satoh et al. (1997) claims that antioxidative efficacy of Trolox (38) surpasses that of α-

tocopherol, the most effective of the vitamin E complex. Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) is a hydrophilic vitamin E analog lacking the phytyl tail, with enhanced antioxidant capacity due to its increased cell permeability.

Trolox is a phenolic antioxidant which was originally designed as a food preservative due

to its free radical trapping capability (Davies et al., 1988).

The 75% fraction of extracts from seven species of Pseudopterogorgia were

screened using the FRAP assay in an attempt to make a systematic comparison of the

antioxidant potential of their biologically available secondary metabolites. The assay was

standardized using Trolox, and results were expressed as µM Trolox equivalents per µg

of extract. The standard curve was linear between 5 and 100 μM Trolox (Figure 19).

Values are reported as µM of Trolox per µg of extract.

The experimental results were expressed as mean ± standard error of mean (SEM)

of four replicates. The data were subjected to one way analysis of variance (ANOVA;

Table A2) and differences between samples were determined by Tukey’s HSD (P< 0.01)

test using the Graphpad Prism program (Table 4; Figure 20). Intra-species variation was not statistically significant (p<0.01, Tukey’s HSD) except for Pseudopterogorgia rigida, which has two different chemical profile, and the Key Largo sample of elisabethae with significantly greater activity than any of the other extracts screened (Table 4; Figure 18).

It is possible that the differences seen in activity within the P. rigida samples may in fact be a result of misidentification of these. The two rigida samples with activity like

74 elisabethae did in fact differ morphologically from most of the other rigida samples

collected. They had a strong acetic acid- like smell and turned the plastic storage bag

Trolox standard curve

1.6 y = 0.0149x + 0.023 R2 = 0.9997 1.4

1.2

1

0.8

0.6 absorbance (593nm)

0.4

0.2

0 0 20 40 60 80 100 120 M Trolox

Figure 19. Example of a standard curve generate using concentrations of Trolox from 5-100 µM in a FRAP assay.

yellow. While sclerite analysis did not differentiate them from other rigida specimen, it is quite possible that they are a different species. In his 1961 monograph, The Shallow- water Octocorallia of the West Indian Region, F.M. Bayer wrote, “It has been shown in the foregoing pages that the characters upon which the classification of octocorals depends – i.e., the ramification and general form of the entire colony, the size, shape and color of the spicules, and the development of the anthocoidial armature in the polyps – are subject to a degree of variation and intergradation that imposes an uncomfortably

large element of doubt upon the resultant system.” We have found several populations in

the Bahamas in which numerous individuals do not readily fit the Pseudopterogorgia

spp. descriptions of Bayer (1961).

75 Table 4. FRAP assay of the intermediate polarity (75%) fraction of seven species of Pseudopterogorgia. Values given are µM of Trolox / µg extract, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

species collection site µM/µg equiv acerosaa Cross Harbor 0.29 ± 0.07 acerosaa Sandy Point 0.12 ± 0.03 bipinnatab Cross Harbor (60ft) 1.34 ± 0.12 bipinnatab,f Cross Harbor (80 ft) 0.93 ± 0.04 elisabethaec Litlle San Salvador 1.92 ± 0.19 elisabethaec Bimini 1.88 ± 0.23 elisabethaec Cross Harbor 1.90 ± 0.27 elisabethaec Burrows (55 ft) 1.95 ± 0.23 elisabethaec Long Rock 1.51± 0.18 elisabethaec San Salvador 1.62 ± 0.15 elisabethaec Cat Island 1.44 ± 0.08 elisabethaec Hog Cay 1.42 ± 0.10 elisabethaed Pickles (KL) 2.13 ± 0.10 elisabethaec Cross Harbor 1.89 ± 0.14 hystrixa Cross Harbor 0.22 ± 0.05 hystrixa Cross Harbor 0.23 ± 0.05 hystrixa Cross Harbor 0.21± 0.01 kallosa AB102 0.34± 0.03 kallosa,f AB102 0.49 ± 0.05 rigidac Long Rock 1.77 ± 0.13 rigidaf Long Rock 0.61± 0.07 rigidaf Sandy Point 0.61± 0.09 rigidac Sandy Point 1.61± 0.08 spa Burrows (20 ft) 0.23 ± 0.03

76 uM Trolox/ug extract 0.00 0.50 1.0 1.5 2.00 2.50 significantly differentmeans( Figure 20. represent µ 0 0

a acerosa Cross Harbor M Trolox/µg extract. a FRAP assayof75%fractionssevenspeciesPseudopterogorgia. acerosa Sandy Point

bipinnata Cross Harbor (60ft) b

bipinnata Cross Harbor (80 ft) f b, c elisabethae Little San Salvador c elisabethae Bimini P c <0.01), Tukey’sHSD.

Mean ofn=4±standarddeviat elisabethae Cross Harbor c elisabethae Burrows (55 ft)

elisabethae Long Rock c

elisabethae San Salvador c 77 elisabethae Cat Island c

elisabethae Hog Cay c d elisabethae Pickles (KL) c elisabethae Cross Harbor aa

ion. Different letters indicate ion. Differentlettersindicate hystrix Cross Harbor

hystrix Cross Harbor

hystrix Cross Harbor aa

kallos AB102 a ,

kallos AB102 f c rigida Long Rock Values Values rigida Long Rock f f rigida Sandy Point c rigida Sandy Point a sp Burrows (20 ft)

There is a lot of inter-species variation. The least and the most potent extracts

show a nearly twenty-fold difference in activity. P. hystrix, which had the lowest overall

activity, is noted by Bayer (1961) to be a deep water specialist, and we have only

observed this species at the deepest site in the region, a ridge along a steep wall that

begins at approximately 28 m. Additionally, unlike the extracts of all the other species in

genus, no terpenoid chemistry was apparent in the 1H-NMR analysis of the 75% fraction

of P. hystrix. Interestingly, extracts of P. acerosa and P. kallos, two of the most common

species on the shallowest reefs in our survey (between 5 and 10 m), had activity that did

not differ significantly from that of the hystrix extract.

To further investigate the antioxidant potential of these extracts, they were

subjected to HPLC analysis. Briefly, 5 mg of each 75% fraction was subjected to semi-

preparative HPLC separation on a polymeric PRP-1 column. We are using an

evaporative light scattering detector (ELSD) to estimate the quantity of compounds

present in the fraction. In addition to HPLC data the medium polar fraction is also

subjected to 1H NMR and 1H-1H COSY to allow the identification of compound classes present in the fraction. The HPLC is equipped with a fraction collector, which was program to collect everything eluted from the column into 96-well plates. These plates were then dried in vacuo and reconstituted with 200 µL of 100% MeOH per well, 10% of which was transferred to 96-well microtitre plates with FRAP reagent and assayed for activity. In this fashion, activity can be correlated with chromatographic peaks, and active components can be rapidly identified for further study (Figure 21, 22). Activity for this assay is expressed as FeSO4 µM equivalents per well.

78

BA07-019 P. rigida BA06-068 P. acerosa 800 800

700

700 600

600 500

500 400

400

uM equivalents uM 300 4 uM equivalents uM 4

300 FeSO FeSO 200

200 100

100 0 1 4 7 101316192225283134374043464952555861646770737679828588919497100103106109112115118121124127130

0 -100 1 4 7 101316192225283134374043464952555861646770737679828588919497100103106109112115118121124127130 vial # P. acerosa vial # P. rigida

BA07-025 P kallos

1400

BA06-052 P bipinnata

1400 1200

1200 1000

1000 800

800 uM equivalents uM 4 600 uM equivalents 4 600 FeSO FeSO 400 400

200 200

0 0 1 5 9 13172125293337414549535761656973778185899397101105109113117121 1 5 9 13172125293337414549535761656973778185899397101105109113117121125129 vial# P. bipinnata P. kallos vial #

Figure 21. comparison of chromatograms and graphical representations of FRAP analysis of 96-well plate collections of HPLC separation of 75% fraction.

79 BA07-002 Gorda Rock 40 ft KL06-087 Pickles Reef 55 ft 3000

3000

2500

2500

2000

2000

1500 uM equivalents uM

1500 4 uM uM equivalents 4 FeSO 1000 FeSO 1000

500

500

0

0 3 6 9 2 8 4 0 3 9 5 4 0 6 5 1 0 0 1 15 1 21 2 27 3 3 36 3 42 4 48 51 5 57 6 63 6 69 72 7 78 8 84 87 9 93

0 3 6 9 5 1 0 6 2 8 1 7 3 9 5 4 0 vial number 12 1 18 2 24 27 3 33 3 39 4 45 4 5 54 5 60 6 66 6 72 7 78 81 8 87 9 93 P. elisabethae Pickles Reefvial # PKL chemotype P. elisabethae Gorda Rock LBB chemotype

BA99-637 Hog Cay BA06-036 Burrows 55ft

3000 3000.00

2500.00 2500

2000.00 2000

1500.00 1500 uM equivalents uM 4

FeSO 1000.00 1000

500.00 500

0.00 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78 81 84 87 90 93 vial number

P. elisabethae Burrows BSS chemotype P. elisabethae Hog Cay ESB chemotype

Figure 22. FRAP assay of 96-well plate collection from HPLC of 75% fraction. For each of four of the chemotypes one chromatogram, corresponding FRAP plate and graph of FRAP values for each well. FRAP values expressed in µM equivalent FeSO4.

80 For each of the fractions analyzed in this way, FRAP activity was correlated to specific peaks on the chromatogram. In some cases, the identity of the active compounds could be assumed based on retention times, research previously done by this investigator

(1H-NMR of 75% fraction) and previously published data. In the case of P. rigida, the active compound is the previously characterized sesquiterpene (-)-curcuphenol (McEnroe and Fenical, 1978). The active compounds from the 75% fraction of P. bipinnata are kallolide A and its acetate and (Figure 6; structures 14, 15) and the bipinnatins, (figure 5; structure 10-13). It is important to note Sanchez and co-workers have shown, using

ITS1–5.8S-ITS2 phylogenetic analysis, evidence for considering the species P. kallos

(Bielschowsky) as the shallow-most morphotype of P. bipinnata from exposed environments (Sánchez et al., 2007). Additionally the presence of the same metabolites within these organisms supports the theory that P. bipinnata and P. kallos belong to the same species. Yet, the activity of the 75% fractions from specimen of these two species have significantly different activity (p< 0.01, Tukey’s HSD; Figure 16), and we have found that the concentration of active secondary metabolites is considerably greater in bipinnata (Data not shown).

The 75% fractions of P. elisabethae had significantly more antioxidant potential than extracts of the other species in the genus (with the exception of P. rigida) and since the activity in the fractions of P. elisabethae was localized to peaks corresponding to the pseudopterosins, these were further purified for additional screening.

Pseudopterosins were purified by chromatographic separation as follows. Ten to

30 mg of the 75% fraction was injected on a Beckman HPLC with a preparative scale C-

18 column and eluted with a linear gradient of acetonitrile and water (70:30 ACN: H2O

81 to 100% ACN). The identity and purity of each compound was confirmed by UV, CD,

MS, 1H, and 13C NMR spectroscopy (Appendix), and by comparison of our spectral data

with those previously reported (Look et al., 1986; Look and Fenical, 1987; Roussis et al.,

1990; Puyana et al., 2004).

All compounds were assayed in quadruplicate at a concentration of 10µM. Values

reported are µM Trolox per µM of pseudopterosin. The experimental results are

expressed as mean ± standard error of mean (SEM) of four replicates. The data were

subjected to one way analysis of variance (ANOVA, table A3) and differences between

samples were determined by Tukey’s HSD (P< 0.01) test using GraphPad Prism (Table

5; Figure 23).

Table 5. FRAP assay of pure pseudopterosins and related molecules isolated from Pseudopterogorgia elisabethae Values given are µM of Trolox / µg extract, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

pseudopterosin Aa 1.056 ± 0.090 pseudopterosin Ba 0.994 ± 0.022 pseudopterosin Ca 1.080 ± 0.023 pseudopterosin Da 1.038 ± 0.011 ps A-D aglycone b 0.698 ± 0.046 ps Ga,d 1.167 ± 0.017 ps I a,d 1.190 ± 0.044 ps J a,d 1.170 ± 0.054 ps K a 1.123 ± 0.046 ps K 2' acetate a 0.984 ± 0.036 ps K 4' acetate a 1.065 ± 0.028 iso-A a 0.941 ± 0.044 iso-B/iso-C a,d 1.118 ± 0.052 iso-D b 0.595 ± 0.023 ps G-J aglycone d 1.311 ± 0.058

82

1.600 d 1.400 a a a a b a,d a,d a,d a a a a a b 1.200

1.000

0.800 M pseudopterosin M pseudopterosin 

0.600 M Trolox/  0.400

0.200

0.000 A pseudopterosin B pseudopterosin C pseudopterosin D pseudopterosin ps A-D aglycone G pseudopterosin I pseudopterosins J pseudopterosins K pseudopterosin ps K 2' acetate ps K 4' acetate iso-A iso-B/iso-C iso-D ps G-J aglycone

Figure 23. FRAP assay pseudopterosins and related molecules from Pseudopterogorgia elisabethae throughout the study region. Values represent µM Trolox/µM pseudopterosin. Mean of n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

Antioxidant activity did not vary widely among the suite of pseudopterosins

tested. For twelve of the fifteen compounds tested, antioxidant potential appears to be

equal to or greater than that of Trolox on a µM/µM basis. Slight, but statistically

significant (Tukey’s HSD, P < 0.01) differences were observed between the ps G-J series

and the other pseudopterosins, and the aglycone of this series has the best activity. The ps

A-D aglycone and iso-D displayed significantly less activity than the other compounds,

but when the ps A-D aglycone was re-evaluted by 1H-NMR, it was evident that the

molecule had decomposed to an ~ 50:50 mix of the aglycone and a di-ketone derivative.

We would expect that the conversion of the catechol to the di-ketone moiety would cause

83 the loss of antioxidant function. It is not clear why iso-D was significantly less active than the other compounds, though it is not unlikely that this difference may be the result of a quantitaion or experimental error.

Phenolics are diverse secondary metabolites abundant in plant tissues (reviewed in Grace and Logan, 2000). Polyphenols possess ideal structural chemistry for free radical scavenging activity and have been shown to be more effective antioxidants in vitro than tocopherols and ascorbate. Antioxidant properties of polyphenols arise from their high reactivity as Hydrogen or electron donors, and from the ability of the polyphenol-derived radical to perform the chain-breaking function of stabilizing and delocalizing the unpaired electron (Blokhina et al., 2003). Trolox (38; Figure 24) is a potent water-soluble antioxidant in biological fluids. It scavenges reactive oxygen and may, consequently, prevent oxidative damages to important biological macromolecules such as DNA, lipids and proteins (Satoh et al., 1997).

O

O OH

HO

TM 38 Trolox

Figure 24. Structure of Trolox, a synthetic vitamin E analog with potent antioxidant activity.

84 Phenolic diterpenes from sage (Salvia officinalis L.) such as carnosic acid (39) and carnosol (40; Figure 25) have been shown to possess antioxidant and anti- inflammatory activities. Carnosic acid is a diterpene that displays high antioxidant activity and which protects biological membranes from lipid peroxidation (Aruoma et al.,

1992; Haraguchi et al., 1995). The antioxidant activity of carnosic acid is due to the two hydroxyl groups found in ortho-position at C11 and C12 of the molecule (Aruoma et al.,

1992). The same mechanism of antioxidative protection by carnosic acid applies in rosemary and sage. Further more, it has been demonstrated that it is a complete set of biosynthetically related antioxidant molecules (and not a single mechanism) that is responsible for alleviating oxidative stress and reducing oxidative damage in species of the plant genera Rosmarinus, Lepechinia, and Salvia (Munné-Bosch and Alegre., 2001).

Figure 25. Structure of carnosic acid and carnosol, phenolic diterpenes from terrestrial plants with potent antioxidant activity

As a protective response against damage from excessive UV-B radiation, plants enhance the biosynthesis of UV-B absorbing and antioxidant phenolic compounds (Körner, 1999).

The induction of enzymes involved in flavonoid biosynthesis with experimentally

85 supplemented UV radiation has been demonstrated (Jaakola and Määttä-Riihinen, 2004).

Previous studies on altitudinal variation of phenolic compounds in different wild and

cultivated taxa from the Asteraceae suggested that factors related to altitude of the growing site are causing significant shifts in the quantity of secondary metabolites in

flowering heads (Zidorn and Stuppner 2001; Zidorn et al. 2005; Spitaler et al. 2006).

Spitaler and coworkers (2008) found an increase of caffeic acid derivatives in flowering heads of A. montana in response to environmental factors related to the altitude of the growing site. It is well established that enhanced UV-B radiation indirectly causes damage to plants by inducing the formation of free radicals, which subsequently target lipids, proteins, carbohydrates, and nucleic acids (Blokhina et al., 2003). It was also demonstrated that phenolic compounds play a vital role in the hydrogen peroxide scavenging system of plants, which besides phenolics comprises peroxidase, ascorbic acid, and glutathione (Takahama and Oniki, 1997).

Many studies have examined the pharmacological potential of the pseudopterosins. They display strong anti-inflammatory and analgesic activity. These diterpene glycosides had potencies superior to those of existing drugs such as indomethacin in mouse ear models (Look et al., 1986). These compounds appear to act by a novel mechanism of action; they are not active against PLA2, cyclooxygenase, and

cytokine release, or as regulators of adhesion molecules. Evidence suggests the

pseudopterosins block eicosanoid release rather than biosynthesis in murine macrophages. Purified pseudopterosins (pseudopterosin E) have been commercialized for use in cosmetics (Bruckner, 2002). They have also be show to exhibit antituberculosis activity. Typical values for growth inhibition of Mycobacterium tuberculosis range

86 between 20 and 60%. However, some of these marine diterpenoids show a very high

growth inhibition rate (Rodríguez et al., 2000). In biological screenings pseudopterosins and related molecules show in vitro cancer cell cytotoxicity in the NCI’s 60 cell-line

tumor panel (Rodríguez et al., 1999; Rodríguez et al., 2000).

Considerably less is know about the ecological roles of these compounds. Thorton

and Kerr (2002) found significantly higher concentrations of pseudopterosins in exposed

to experimentally elevated levels of feeding by the mollusk Cyphoma gibbosum

indicating the inducibility of pseudopterosin biosynthesis by predation. Similar results to

C. gibbosum predation were obtained in response to decreased levels of UV/VIS

radiation (Thorton and Kerr, 2002). It has been shown that pseudopterosins A-D (17-20)

deter feeding by a natural assemblage of reef fishes, but that the pseudopterosin A-D

aglycone (26) acts as a feeding attractant (Mukherjee, 2005). Ferns and co-workers

(2005) have suggested that the aglycone may be the final product of the pseudopterosin

biosynthetic pathway. The results presented here indicate that ps G-J aglycone has the

highest anti-oxidant potential. It is quite possible that these molecules serve both as

predator deterrence (in the glycosylated form), and as antioxidants (both glycosylated and the more active aglycone form). Optimization of defenses can lead to multiple roles for secondary metabolites. If costs of synthesizing and maintaining chemical defenses are high then utilizing a compound for multiple purposes should increase the overall fitness of an organism relative to producing several compounds for several purposes. The potent and varied biological activities of marine plant and animal extracts suggest that some compounds may fulfill several functions for their host organisms. However, demonstration that compounds are involved in multiple interactions requires that we

87 know the identity of compounds, and that we test purified compounds rather than whole organisms or crude extracts. Therefore, rarely have discrete metabolites been tested for their capacity to fulfill multiple ecological roles.

In a study done by Thorton and Kerr (2002) he blocking of UV/visible radiation increased the pseudopterosin content by approximately 100% in the treatment groups.

While the authors remark that ecological significance of this is not immediately clear, they imply that it suggests the involvement, either directly or indirectly, of a photosynthetic organism in pseudopterosin biosynthesis. If the pseudopterosins are being used as antioxidants in what would probably be a non-recycled fashion, we would not be surprised to find lower concentrations of these in the tissues of organisms exposed to higher light regimes.

The work presented in this chapter represents the first time secondary metabolites from octocorals have been examined for antioxidant potential. The results strongly suggest that secondary metabolites from organisms in the genus Pseudopterogorgia may serve as antioxidants. Goulet and co-workers (2008) assessed the relative sensitivities of zooxanthellate octocorals to thermal stress across numerous reefs in the central GBR system during the 1998 sea surface heating event. Video transects showed that across all taxa, on average 43% of inshore colonies and 21% of offshore colonies, were partially or totally bleached (Goulet et al., 2008). The percentage of bleached colonies differed substantially among taxa during the height of the bleaching event. For example at the height of the bleaching event <10% of colonies of Briareum spp., Rhytisma fulvum,

Lemnalia spp., and Paralemnalia spp. were partially or totally bleached. Samples of bleached and unbleached tissues were collected for genetic analyses of resident

88 Symbiodinium populations, and symbiont genetic identity was not found to be the cause of variability in bleaching response within and between soft coral species. The authors of

the study conclude that differential tolerance to stress among octocoral species

irrespective of the Symbiodinium they host, may explain differences in bleaching (Goulet

et al., 2008). Soft corals of the genera Lemnalia, Paralemnalia, and are a rich source of sesquiterpenoids, and the genus Briareum is a prolific source of diterpenoids.

Perhaps it is the isoprenoid chemistry that confers the organisms in these genera a reduce susceptibility to bleaching. Results presented here support this hypothesis as they provide a potential mechanism to effect this variability; differing secondary metabolite chemistry.

If the increasing incidence of bleaching events continues, the relative insensitivity of octocorals to thermal stress may have important consequences for the future of reef ecosystems.

89 CHAPTER 4

In vivo antioxidant activity of extracts and pure compounds from Pseudopterogorgia

sps.

4.1 Photosynthesis and oxidative stress

Corals containing symbiotic dinoflagellates (zooxanthellae) of the genus

Symbiodinium are dependent upon light energy to power photosynthesis. The

photochemical apparatus within zooxanthellae is similar to those found in other oxygen-

evolving photosynthetic algae and plants. There are two photosystems involved, called

Photosystem I (PS I) and Photosystem II (PS II). PS II is associated with the splitting of

water molecules to form molecular oxygen. PS I involves transfer of energy to the Calvin

Cycle, where inorganic carbon is converted to simple sugar. Photosynthesis begins with

Photosystem II. If PS II fails, the electron flow from PS II to PS I stops, and the entire

photosynthetic process grinds to a halt. Light-absorbing pigments called photopigments

harvest light energy. Chlorophylls (chlorophyll a and chlorophyll c2) are the most

recognized photopigments of zooxanthellae, but others are involved as well, and these are

called accessory (or antennae) pigments. In corals, accessory pigments include peridinin and beta-carotene. Chlorophylls and accessory pigments can channel the energy they harvest to both photosystems, although it is possible for an accessory pigment (such as

beta-carotene, peridinin, etc.) to transfer its collected energy almost exclusively to one

photosystem.

90 A small proportion of excitation energy is dissipated by the emission of

fluorescence, stemming almost exclusively from chlorophyll a of photosystem II.

Fluorescence emission competes with two other de-excitation processes that deactivate

the excited chlorophyll states. These processes reduce (or quench) the amount of

fluorescence, and are referred to as photochemical quenching (qP) and non-

photochemical quenching (qN). Photochemical quenching reflects useful photochemistry

(i.e. assimilatory or non-assimilatory electron flow), and depends upon the presence of

oxidized Qa (a quinone-type primary electron acceptor in photosystem II). When Qa is

oxidized, it can accept electrons from the photosystem II reaction centre, and pass these

along the photosynthetic electron transport chain. The result is the oxidation of water,

oxygen evolution, the reduction of NADP+ to NADPH, membrane proton transport, ATP

synthesis and eventually to the reduction of CO2 to carbohydrate in the dark reactions of

photosynthesis (Calvin cycle). The other means by which excited chlorophyll states can

be deactivated is non-photochemical quenching, which reflects photoprotective

dissipation of excess absorbed energy as heat in the light-harvesting antennae (Demmig-

Adams, 1990; Horton and Ruban, 1994).

Active oxygen species are produced by a variety of metabolic processes; however

the major source of active oxygen species in plant tissues is the photosynthetic electron

transport system (Asada 1994). In photosynthesis, molecular oxygen is produced from

the oxidation of water by the photosynthetic electron transport chain (Foyer et al. 1994).

Oxygen can also be used as an electron acceptor, a reaction that results in the formation

1 2- of singlet oxygen ( O2) and superoxide radicals (O ) (Fridovich 1986; Foyer et al. 1994).

Singlet oxygen is highly destructive as it reacts with most biological molecules (Knox

91 and Dodge 1985). Superoxide is also highly toxic with several cellular targets, however

it’s primary impact is an indirect one, as it gives rise to more powerful oxidants such as hydrogen peroxide (H2O2) and the hydroxyl ion (HO) (Fridovich, 1986). The principal

sites of damage from these toxic oxygen species include the primary carboxylating

enzyme, Rubisco, and Photosystem II, in particular, the D1 protein of PSII, which

appears to have very little protection against oxidative damage (Asada and Takahashi

1987; Richter et al., 1990; Tschiersch and Ohmann, 1993). Damage to the D1 protein caused by reactive oxygen is irreversible, and can be corrected only by the enhanced synthesis and replacement of the protein (Long et al., 1994).

Symbiotic cnidarians and their zooxanthellae do produce hydroxyl radicals upon illumination (Dykens et al. 1992). Increased activities of antioxidant enzymes in the

Symbiodinium sp. appears to be insufficient to scavenge the increase in the cellular flux of superoxide radicals and hydrogen peroxide produced during exposure to UV radiation and elevated temperatures (Lesser, 1996). Additionally, Lesser (1997) has shown that antioxidants prevent the coral from bleaching and affect the mechanism of symbiont loss from the coral host.

4.2 Detection of intracellular ROS using 2’,7’-dichlorodihydrofluorescein diacetate

Reactive oxygen species (ROS) are encountered by all aerobic organisms, with oxidative proclivity being especially high at electron rich areas such as metabolic or photosynthetic sites. Marine organisms are exposed to particularly high levels of ROS through a combination of photosynthesis, symbiont oxygen production, and light

92 intensities leading to UV-induced free radical production. The conjecture that organisms highly exposed to ROS will have effective antioxidant mechanisms has certainly not disappointed natural product chemists. Many species contain powerful plant-like—or completely novel— antioxidant compounds (Dunlap and Walter, 2003). Researchers have recently begun to look at the antioxidant effects of natural products in living systems. In a recent study, Takamatsu and coworkers (2003) investigated antioxidant activity of over one hundred purified marine natural product compounds using a 2’,7’- dichlorodihydrofluorescein diacetate (DCFH-DA) cell-based method to directly examine the ability of natural products to penetrate living human carcinoma cells (HL-60) and inhibit reactive oxygen species (ROS)-catalyzed oxidation. Their results clearly demonstrate the potential of marine natural products to act as potent antioxidants.

Screening of extracts and pure compounds of seven species of the genus

Pseudopterogorgia using the FRAP assay (as described in Chapter 3) indicate that these have promising antioxidant potential. Often it is necessary to obtain a variety of information about antioxidant potential other than what can be concluded from the FRAP assay alone. Cell-based assays in particular can be used to gain appreciable evidence of the potential in vivo antioxidant effectiveness of a compound. To examine the potential ecological significance of these results, two assays were developed employing cultured

Symbiodinium spp cells.

The in vivo antioxidant activity of the intermediately polar fractions of seven species of the genus Pseudopterogorgia was tested in a zooxanthellae based assays. This was accomplished using cultured zooxanthellae and the oxidative stress sensitive dye

93 DCFH-DA in a micro-well plate. DCFH-DA diffuses into the cell where cellular

esterases cleave the diacetate moiety to form the more polar DCFH, which is trapped

within the cell. Oxygen radicals oxidize the intracellular DCFH to the fluorescent DCF.

The level of fluorescence measured upon excitation is proportional to the level of

oxidation. Antioxidants prevent oxidation of DCFH and reduce the formation of DCF.

Fluorescent technology has made it possible to evaluate antioxidants in living

cells using specific probes such as 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-

DA). This cell-based fluorescent method is useful to directly examine the ability of

natural products to penetrate cell membranes and inhibit ROS. Due to the indiscriminate

nature of DCFH, which can be oxidized by various ROS and not just H2O2, the increase

of intracellular DCF fluorescence does not necessarily reflect the levels of ROS directly,

but rather an overall oxidative stress index (OS) in cells (Wang and Joseph, 1999).

Quantifying cellular OS by the DCF assay using a fluorescent microplate reader is an easy and efficient method with low variability which can be used to quantify the potency of pro-oxidants or can be adapted to evaluate the efficacy of antioxidants against ROS in various cell lines (Wang and Joseph, 1999).

The formation of the fluorescent DCF from DCFH involves a complex process that occurs in close interaction with the superoxide radical and hydrogen peroxide. These two species react to form hydroxyl radicals in the Fe-catalysed Fenton (Haber-Weiss) reaction. DCFH reacts with hydroxyl radicals to for the DCF radical that reacts with oxygen and produces the green-fluorescent DCF (Zu et al., 1994). Glutathione suppresses

DCF formation as it reconstitues the DCF radical into DCFH (Zu et al., 1996), and

94 antioxidants such as mannitol scavenge active oxygen species in competition with DCFH

(Shen et al., 1997). Trolox works in a similar fashion as mannitol.

An experiment was designed for the assessment of active oxygen production and

the evaluation of the antioxidant activity of the extracts and pure compounds from

Pseudopterogorgia sps. Clade C2 Symbiodinium culture from the lab of Gregory

Schmidt, Department of Plant Biology, University of Georgia were used in this assay. We chose this particular culture because results of a previous study suggest that they are relatively more sensitive to the synergistic effects of thermal and irradiance stress than other clades (Berkelmans and van Oppen, 2006). Zooxanthellae cells were incubated in

10 μM DCFA-DA probe for 30 min with constant agitation. Excess probe was than washed away and the cells were resuspended in seawater. A total of 190 μL cell suspension was added to each well of black-bottom 96-well microtiter plates. Extracts

(50 µg each dissolved in methanol) were added to wells in quadruplicate, control wells had either methanol only or cells incubated with the probe only. The plates were maintain in a water bath at 32°C and exposed to 180 µM of light. A control plate with the same volume of DCFH-DA treated cells per well as the experimental plates was maintained at room temperature in ambient light. Fluorescent intensity was recorded using a monochromatic spectrophotometer set at excitation wavelength 485 and emission wavelength 538. The level of fluorescence measured upon excitation is proportional to the level of oxidation. Antioxidants prevent oxidation of DCFH and reduce the formation of DCF. Readings were taken once an hour for four hours, and plates were dark acclimated for twenty minutes prior to each reading. Fluorescence in control cells that were exposed to the experimental thermal and irradiance stress was 6.63 times higher on

95 average than those that were maintained at 26º C in ambient light (60µM). This serves to validate that the method employed to induce oxidative stress in the zooxanthellae was successful.

Raw data was converted to percent inhibition versus the solvent only control cells using the following equation:

[(Fc – Ft ) / Fc ] x 100

Were Fc is the average of the solvent only control wells and Ft is the average for a given treatment (extract or compound). It is important to note that the addition of the solvent

(MeOH) did cause a slight but not statistically significant reduction in fluorescence, thus the solvent only controls have been used to calculate the percent reduction.

Table 6. DCFH-DA assay of the intermediate polarity (75%) fraction of seven species of Pseudopterogorgia. Values given are % reduction in DCF fluorescence versus MeOH control, n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

acerosa Bu a 5.28 ± 3.93 acerosa SP a 4.41 ± 3.57 bipinnata CH b 21.26 ± 2.96 bipinnata CH b 19.99 ± 3.51 elisabethae Bi b,c 31.51 ± 1.48 elisabethae Bu b,c 28.36 ± 2.08 elisabethae CH b,c 25.69 ± 3.33 elisabethae KL c 34.15 ± 1.70 elisabethae LR b,c 26.24 ± 4.43 elisabethae SP b,c 27.02 ± 2.88 elisabethae SS b,c 28.62 ± 2.12 hystrix CH a 5.73 ± 1.55 kallos SP a,d 11.56 ± 1.87 rigida CH a,d 15.55 ± 2.77 rigida LR c 30.94 ± 2.86 sp CH a 7.15 ± 2.08

96 DCFH-DA zooxanthellae assay

40.00

35.00

30.00

25.00

20.00 a a a 15.00 a % inhibition vs control vs inhibition % b,c b,c b,c b,c b,c c b,c

10.00 b b sp CH sp a,d c a,d hystrix CH acerosa Bu

5.00 acerosa SP kallos SP SP kallos CH rigida rigida LR elisabethae Bi, elisabethae SS bipinnata CH bipinnata CH bipinnata elisabethae Bu elisabethae CH elisabethae KL elisabethae LR 0.00 elisabethae SP

Figure 26. Results of the zooxanthellae-based DCFH-DA assay given as percent fluorescence inhibition versus control cells (MeOH only). n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

The experimental results were expressed as mean ± standard error of mean (SEM) of four replicates. The data were subjected to one way analysis of variance (ANOVA) and differences between samples were determined by Tukey’s HSD (P< 0.01) test using the Graphpad Prism program (Table 6; Figure 26). Means were significantly different

(ANOVA P< 0.05; see Appendix). Intra-species variation was not statistically significant

(p<0.01, Tukey’s HSD) except for Pseudopterogorgia rigida, which has two different chemical profiles, one of which was as active as the P elisabethae fractions. The overall activity in this assay correlates well with the results of the FRAP assay presented in

97 Chapter 3. We can conclude that extracts not only exhibit antioxidant potential in a

solution-based assay but can also be taken up by living cells and maintain their activity.

Pure pseudopterosins were screened in this assay following the same protocol as

detailed above, except each of the compounds were added to the wells so that the final

concentration of each was 20 µM. Trolox was used as a positive control, also at a 20µM

final concentration. The data were subjected to one way analysis of variance (ANOVA,

means significantly different, p<0.05; see Appendix) and differences between samples

were determined by Tukey’s HSD (P< 0.01) test using GraphPad Prism (Table 7; Figure

27). Antioxidant activity did not vary widely among the suite of pseudopterosins tested.

For ten of the fifteen compounds tested, activity appears to be equal to that of Trolox at

the same concentration. Ps A-D aglycone displayed significantly less activity than the

Table 7. FRAP assay of pure pseudopterosins and related molecules isolated from Pseudopterogorgia elisabethae, given as percent fluorescence inhibition versus control cells (MeOH only) n=4 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

pseudopterosin A a,b 44.951 ± 2.284 pseudopterosin B a,c 47.454 ± 0.916 pseudopterosin C a,c 47.793 ± 0.682 pseudopterosin D a,b 44.538 ± 1.626 ps A-D aglycone b 38.340 ± 3.495 pseudopterosin G a,c 46.460 ± 1.400 pseudopterosin I a,b 44.801 ± 0.791 pseudopterosin J a,b 45.026 ± 3.262 pseudopterosin K a,c 45.928 ± 1.218 pseudopterosin K 2' acetate a,c 48.082 ± 1.673 pseudopterosin K 4' acetate a,c 48.136 ± 3.130 iso-A a,c 45.460 ± 2.030 iso-B/iso-C a,c 45.891 ± 2.095 iso D a,c 46.878 ± 0.972 ps g-J aglycone a,c 51.451 ± 0.614 TROLOX c 52.686 ± 2.287

98 positive control and ten of the fourteen other compounds, but when the ps A-D aglycone

was re-evaluated by 1H-NMR, it was evident that the molecule had decomposed to an ~

50:50 mix of the aglycone and a di-ketone derivative. We would expect that the conversion of the catechol to the di-ketone moiety would cause the loss of antioxidant

function. It appears evident from the results of this experiment that at least some portion

of the bioactive components from these intermediately polar fractions is able to penetrate the Symbiodinium sp cells. Additionally, these fractions of the extracts from the seven species of Pseudopterogorgia and pure pseudopterosins reduce zooxanthellar intracellular OS as evidence by the reduction in DCF fluorescence. The pseudopterosins appear to be equally as efficient in reducing OS in zooxanthellae as Trolox at the 20µM

concentration.

60.000

50.000

40.000

30.000 % inhibition versus control versus inhibition % 20.000

10.000 pseudopterosin A a,b pseudopterosin B a,c pseudopterosin C a,c pseudopterosin D a,b psA-D aglyconeb pseudopterosin G a,c pseudopterosin I a,b pseudopterosin J a,b pseudopterosin K a,c pseudopterosin K2' acetate a,c pseudopterosin K4' acetate a,c a,c iso-A a,c iso-B/iso-C a,c D iso psg-J aglycone a,c TROLOX c 0.000

Figure 27. Results of the zooxanthellae-based DCFH-DA assay given as percent fluorescence inhibition versus control cells (MeOH only). N=3 ± standard deviation. Different letters indicate significantly different means (P <0.01), Tukey’s HSD.

99 Oxidative stress within the host tissues can occur as a consequence of oxidative

stress in the algal symbiont because hydrogen peroxide can diffuse through biological membranes into host tissues (Asada and Takahashi 1987). It is known that Trolox exerts its antioxidant effect through its capacity to scavenge free radicals, but it is not possible to conclude that this is the nature of the activity of the pseudopterosins in vivo. In any case, it is clear that the antioxidant activity of the secondary metabolites of species from the genus Pseudopterogorgia would have adaptive value, particularly given the increasing incidence of El Niño/Southern Oscillation events and associated increase in prevalence and severity of coral bleaching.

4.3 Detection of photoinhibition using PAM fluorometry

Photoinhibition has been defined as light-dependent inhibition of photosynthesis

(Powles, 1984; Greer and Laing, 1991). It can be described as the decreased capacity of a photosystem to capture and process photons (Long et al., 1994; Osmond, 1994), and is characterized by the accumulation of photochemically inactive PSII reaction centers

(Krause, 1994). This typically results in a decrease in the overall photosynthetic rate

(Richter et al., 1990). Photoinhibition is one of the daily challenges faced by most photosynthetic organisms (Hoegh-Guldberg and Jones, 1999). Photoinhibition occurs when the utilization of energy by the fixation of CO2 is exceeded by the amount of

incoming light energy (Greer and Laing, 1991). Reduction in photosynthetic electron

transport combined with continued high absorption of excitation energy leads to

inactivation of, or damage to, PSII from the production of toxic oxygen species (Osmond,

1994; Lesser, 1996). It has been proposed that the reduction of PSII efficiency may act as

100 a protective mechanism to prevent damage to the photosynthetic apparatus, rather than

indicating the degradation of the D1 protein of PSII. Therefore it is important to

discriminate between the inactivation of PSII as a reversible downregulation of

photosynthesis, and the irreversible effects of photodamage (Critchley and Russell,

1994).

The source of chlorophyll fluorescence in algae and plants is the antennae

pigments of photosystem II. The initial fluorescence, F0, of the samples is a measure of

fluorescence when the reaction centers of photosystem II are fully oxidized. A saturating

pulse of white light causes reduction of the photosystem II reaction centers and

fluorescence increases to a maximal value (Fm). The change in fluorescence from F0 to

Fm (ΔF) denotes the variable fluorescence. The ratio of variable to maximal fluorescence

in a dark-adapted sample (Fv/Fm,) can be correlated to the quantum yield of

photosynthesis and serves as a convenient measure of the maximum potential quantum

yield (Björkman and Demmig, 1987; Schreiber and Neubauer, 1990). Chlorophyll

fluorescence measured in this fashion provides valuable information on the efficiency of

photosynthesis, and fluorescence measurements are now widely used in studies on the

effects of environmental stress. Knowledge of the variation in fluorescence

characteristics among plants grown under non-stress conditions, and of the relationship

between these characteristics and the photon yield in stressed plants is therefore of

considerable importance. Kitajima and Butler (1975) have proposed that the ratio of

variable to maximum fluorescence (Fv/Fm) emitted from photosystem II (PSII) may

serve as a quantitative indicator of the efficiency of the photochemistry of PSII.

101 The endosymbiotic dinoflagellate microalgae (Symbiodinium spp.) located

intracellularly within the tissues of scleractinian corals are very sensitive to elevated

water temperature (Coles and Jokiel, 1978; Glynn and D.Croz ,1990; Iglesias- Prieto et

al., 1992). Several studies have shown that at temperatures of 32°C photosynthesis is

impaired (Iglesias Prieto et al., 1992; Fitt and Warner 1995; Warner et al., 1996; Iglesias-

Prieto, 1997; Lesser, 1997; Jones et al., 1998). This thermal sensitivity of Symbiodinium

spp. Is believed to be the underlying cause of the phenomenon of coral bleaching

(Iglesias-Prieto et al., 1992), and has been investigated by photo-respirometry and

chlorophyll fluorescence techniques. The chlorophyll fluorescence techniques have been

particularly useful because they are rapid and non-invasive, and they allow diagnostic

analyses of the effects of heat stress (see for example Warner et al., 1996; Jones et al.,

1998). Temperature and light stress in corals is readily detectable as a change in the

photosynthetic fitness of the zooxanthellae using non-invasive pulse amplitude

modulation (PAM) fluorometry. The fluorometer uses a range of flashing (pulsed) lights

to measure the photosynthesis. These pulses of light are modulated, and the height (or

amplitude) of the pulse of light governs its intensity.

The ratio of variable fluorescence of variable (Fv) to maximal (Fm) fluorescence

(Fv/Fm) can be quantitately related to the efficiency of photosynthesis. When all reaction

centers are open, the probability of excitation escape is low and thus fluorescence is

minimal (F0). When the reaction centers are become closed, absorbed excitation energy

cannot be directed to photochemistry and fluorescence emitted rises to the maximum

value, Fm. The ratio of quantum yields for fully open and fully closed reaction centers, normalized to Fm is a measure of quantum yield of photochemistry in PS II, and reflects

102 the probability that PS II reaction centers will use available excitation energy for

photochemistry.

Coral bleaching is related to the general phenomenon of photoinhibition. The use

of pulse amplitude modulated (PAM) fluorometry to study heat-stressed a coral has

initiated the identification of the component of the photosynthetic metabolism that fails

when zooxanthellae are exposed to heat stress (Iglesias-Prieto et al., 1992; Fitt and

Warner, 1995; Warner et al., 1996). PAM measures variable fluorescence which is a relative measure of the rate at which PS II can use light to process electrons flowing from the water-splitting reactions of photosynthesis. Bleached corals have low maximum quantum yield (i.e. photochemical efficiency) of the photosystem II (PSII) as measured by the ratio of variable (Fv) to maximal (Fm) fluorescence (Fv/Fm) (Fitt and Warner,

1995; Warner et al., 1996, 1999; Brown et al., 2000; Jones et al., 2000; Bhagooli and

Hidaka, 2002) and display a decline in the D1 reaction center protein (Warner et al.,

1999). Recent studies showed that photoinhibitory damage of zooxanthellae as measured by long-term reduction in photochemical efficiency leads to bleaching (Brown et al.,

2000; Jones and Hoegh- Guldberg, 2001). Corals often bleach first on their upper sunlight-exposed surfaces (Williams and Bunkley- Williams, 1990). This pattern is consistent with manipulative experiments that show that impairment of photosynthesis in corals during heat stress is dependent on both the quality (Fitt and Warner, 1995) and quantity (Jones et al., 1998) of light.

A decrease in Fv/Fm of the symbiotic dinoflagellates in the corals Agaricia

lamarckii, A. agaricites, Montastrea annularis, Siderastrea radians (Fitt and Warner,

1995; Warner et al., 1996) and Stylophora pistillata (Jones et al., 1998) has been recorded

103 in laboratory experiments during heat stress. In these studies, the decrease in Fv /Fm

preceded loss of dinoflagellates from the tissues and bleaching. The temperature at which

photosynthetic dysfunction occurs during manipulative studies of heat stress in corals is

similar to those observed during natural coral bleaching events.

To cope with oxidative stress, plant cells have developed many highly-efficient

defense systems, with both enzyme and non-enzyme constituents. The non-enzymatic

antioxidants are generally small molecules, such as ascorbate and glutathione, that occur

in the aqueous phase (intracellular fluid), and lipophilic antioxidants (tocopherols and

carotenoids) which are active in cell membranes (Noctor and Foyer, 1998; Munne-Bosch

and Alegre, 2002. In a study done to investigate the response of an ascorbate-deficient

Arabidopsis thaliana, mutant vtc1 plants (deficient in ascorbate due to a mutation) were

exposed to short-term increased UV-B. Gao and Zhang (2008) found Fv/Fm decreased

much more in the vtc1 mutant than in the wild type, validating the importance of the non-

enzymatic antioxidant defense. The research of Havaux et al. (2005) shows that vitamin

E protects Arabidopsis thaliana against photoinhibition and photooxidative stress.

Additionally, herbicide-induced blocking of the tocopherol synthesis in the green alga

Chlamydomonas reinhardtii exposed to high light was associated with a loss of

photosystem II (PSII) activity (Trebst et al., 2002). Based on this observation, it was

suggested that -tocopherol could be specifically involved in the maintenance of the PSII

1 function by scavenging O2 produced at the PSII reaction center. In a study by Lesser

(1997) the Caribbean coral Agaricia tenuifolia, was exposed to temperature induced

stress and exposure to full solar radiation. They exhibited an overall decrease in

photosynthetic performance which was followed by bleaching. Exposure of corals to the

104 exogenous antioxidants, Vitamin C (125µM) and mannitol (10mM), known to scavenge

reactive oxygen species during temperature-induced stress, improved maximum

photosynthetic capacity to rates indistinguishable from corals measured at the ambient

temperature of their site of collection and prevented bleaching.

The ability of secondary metabolites isolated from Pseudopterogorgia elisabethae

to delay photoinhibition in stressed zooxanthellae was tested as follows. Samples from a

clade B2 Symbiodinium culture from the temperate western Atlantic were subjected to combined irradiance (light=300µM) and thermal (temperature=34ºC) stress. Maintaining

constant temperature was achieved by placing the sample vial in a jacket with water circulating though a water bath (Figure 28). Pulse-amplitude modulated (PAM) fluorometry was used to characterize the maximum quantum efficiency of PSII (FV/FM).

Samples were dark acclimated for 20 minutes before initial PAM reading. Each sample was then subjected to 14 automated cycles consisting of 35 minutes of light followed by

5 minutes after which a PAM measurement was recorded using a Diving-PAM (Walz,

Germany).

a b

Figure 28. The photograph shows a) the apparatus used for the photoinhibition assay, and b) the Diving PAM fluorometer.

105 0.7 CONTROL, a DMSO, b Ps G-J aglycone 100 uM, c 0.6 ps K 2' ac 50 uM, a ps K 2' ac 75 uM, a

0.5 Ps K 75 uM, a

0.4 Fv/Fm 0.3

0.2

0.1

0 0 100 200 300 400 500 600 time (minutes)

Figure 29. n=3 ± standard deviation. Different letters indicate significantly different means (P <0.05), Tukey’s HSD.

Table 8. Pairwise comparison of treatments; Tukey’s HSD, p<0.05.

Tukey’s Multiple Comparison Test q Significant? P < 0.05? CONTROL vs DMSO 6.20 Yes CONTROL vs Ps G-J Aglycone 100 µM 20.59 Yes CONTROL vs Ps K 75 µM 3.80 No CONTROL vs K 2’ ac 50µM 2.01 No CONTROL vs K 2’ ac 75 µM 2.74 No DMSO vs Ps G-J Aglycone 100 µM 26.79 Yes DMSO vs Ps K 75 µM 10.00 Yes DMSO vs K 2’ ac 50µM 8.21 Yes DMSO vs K 2’ ac 75 µM 8.94 Yes Ps G-J Aglycone 100 µM vs Ps K 75 µM 16.79 Yes Ps G-J Aglycone 100 µM vs K 2’ ac 50µM 18.58 Yes Ps G-J Aglycone 100 µM vs K 2’ ac 75 µM 17.85 Yes Ps K 75 µM vs K 2’ ac 50µM 1.79 No Ps K 75 µM vs K 2’ ac 75 µM 1.06 No K 2’ ac 50µM vs K 2’ ac 75 µM 0.73 No

106 Results were compared to control samples to which no compounds were added

and solvent-only controls. Pairwise comparison of treatments using Tukey’s HSD

indicate that pseudopterosins, and especially the pseudopterosin G-J Aglycone reduce the loss of quantum photosynthetic yield over the nine and a half hour period of the experiment versus the solvent only control (Figure 29, Table 8). Loss of quantum photosynthetic yield for the aglycone treated samples was also significantly less than the control samples without solvent. DMSO also significantly reduced quantum photosynthetic yield when compared to the cells only control. Dimethyl sulphoxide

(DMSO) is commonly used as a solvent for water-insoluble substances applied to

biological material. A very small volume was added to the samples, and thus it is unclear

why these samples differed significantly from the control samples without solvent.

There is a strong suggestion that both coral reef organisms and communities

exhibit acclimative and "adaptive" behaviors and considerable flexibility of response to

environmental change, at time scales of years to decades (Buddemeier and Smith, 1999).

Many compelling examples that confirm the prominence of protein metabolism as a central component of the acclimatization biology in tropical reef corals. For example, a suite of mycosporine-like amino acids (MAAs) screens UV radiation and protects corals from cellular and molecular damage (Jokiel and York, 1982; Gleason and Wellington,

1993). The ability of corals to increase and decrease expression levels of hsp 70, and effectively regulate the activity of antioxidant enzymes clearly reflects the intrinsic biological flexibility of reef corals with respect to their environment (Gates and Edmund,

1999). Reef corals regulate the activity of antioxidant enzymes to ameliorate the deleterious effects of oxygen radicals generated in their tissues in response to small

107 environmental change (Lesser, 1997). Following exposure to elevated temperature and

UV radiation, increased activities of superoxide dismutase (SOD) and catalase (CAT)

have been directly measured in the coral Pocillopora damicornis exposed to differential

flow regimes (Lesser et al., 1994), and in the soft-bodied symbiotic cnidarians

Anthopleura elegantissima (Dykens and Shick, 1984) and Palythoa caribaeorum (Lesser

et al., 1990). Different sides of Goniastrea aspera colonies that were either sun-exposed

or shaded during emersion at low tide contained different levels of host heat shock

proteins and the superoxidase dismutase (Brown et al., 2002).

Differential tolerance to stress among cnidarian species, irrespective of the

Symbiodinium they host, may explain differences in bleaching (Gates and Edmunds,1999;

Loya et al., 2001; Bhagooli and Hidaka, 2004). While the studies referenced above have examined changes in protein metabolism in response to environmental stress, our study

represents the first attempt to examine the relationship between secondary metabolite

chemistry and oxidative stress in symbiotic cnidarians. It has been demonstrated

previously that the addition of exogenous antioxidants to an illuminated culture of

Euglena gracilis not only improved variable fluorescence and photosynthetic

performance, but also decreased the rate of membrane-lipid peroxidation (Tschiersch and

Ohmann, 1993). The addition of exogenous scavengers of oxygen radicals restores a

significant portion of the photosynthetic capacity in cultured zooxanthellae exposed to

UV radiation and elevated temperatures (Lesser, 1996). Lesser (1997) has also shown

that in the coral Agaricia tenufolia, photosynthetic performance returned to pre-stress

rates when the coral are treated with exogenous antioxidants.

108 Our results clearly demonstrate that the bioactive components of extracts from

seven species in the genus Pseudopterogorgia are taken up by Symbiodinium cells.

Furthermore, they reduce the overall oxidative stress index in the stressed cells as

measured using the probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA).

Equivalent micromolar concentrations of ten of the fifteen pseudopterosins tested and the

known antioxidant trolox exhibit the same activity. Thus, we conclude that a potential

ecological role for the diverse and novel secondary compounds by species in the genus

Pseudopterogorgia may be defense against oxidative stress. Previously it has been shown

that the octocoral families Briareidae, Clavularidae, Gorgonidae, and Nephtheidae exhibited low bleaching susceptibilities (Goulet et el., 2008). Soft corals of the genera

Lemnalia, Paralemnalia, and Rhytisma are a rich source of sesquiterpenoids, and the genus Briareum is a prolific source of diterpenoids. Perhaps it is this isoprenoid chemistry that confers the organisms in these genera and the Pseudopterogorgia their reduced susceptibility to bleaching. The experimental evidence that bleaching in corals exposed to elevated temperatures can be prevented by antioxidants (Lesser, 1997), along with the results presented here seem to support this hypothesis.

109 CHAPTER 5

A potential pharmacological role for the antioxidants from Pseudopterogorgia sps.

5.1 Oxidative stress and human diseases

All aerobic organisms are susceptible to oxidative stress simply because semireduced oxygen species, superoxide and hydrogen peroxide, are produced by mitochondria during respiration. However, during pathological conditions, the oxidant / antioxidant balance is altered. Oxidative stress has been implicated in various pathological conditions involving cardiovascular disease, cancer, neurological disorders, diabetes, ischemia/reperfusion, other diseases and aging (Dalle-Donne et al., 2006).

Accumulation of free radical damage as the cause of aging is supported by studies that show increased oxidatively induced protein and lipid damage in pathological conditions of accelerated aging (Stadtman, 2004). A particularly important consequence of free radical damage in many cells, is the peroxidation of polyunsaturated fatty acids (PUFA), which results in the formation of lipid peroxides and aldehydes (Cheeseman and Slater,

1993), and carbonyl modifications of proteins which may affect a variety of cellular functions involving proteins: namely; receptors, signal transduction mechanisms, transport systems and enzymes.

Oxidative stress refers to the cytopathologic consequences of a mismatch between the production of free radicals and the ability of the cell to defend against them.

Oxidative stress can thus occur when the production of free radicals increases, scavenging of free radicals or repair of oxidatively modified macromolecules decreases,

110 or both. This imbalance results in a build-up of oxidatively modified molecules that can cause cellular dysfunction and, for postmitotic cells such as neurons, cell death.

Mitochondrial oxidative metabolism, nitric oxide, phospholipid metabolism, and proteolytic pathways are potential sources of intracellular free radicals. Alterations in free radical defense systems may also contribute to oxidative stress. A net increase in reactive oxygen species can produce damage to lipids, proteins, and DNA and induce necrosis or apoptosis.

Free radicals are normal products of cellular aerobic metabolism (McCord, 1985;

Halliwell and Gutteridge, 1985). Superoxide and hydroxyl species are the predominant cellular free radicals. Hydrogen peroxide and peroxynitrite although not themselves free radicals, contribute importantly to the cellular redox state. Together, these molecules are referred to as reactive oxygen species (ROS). An array of cellular defense systems exist to counterbalance ROS (McCord, 1985; Halliwell and Gutteridge, 1985). These include enzymatic and non-enzymatic antioxidants that lower the steady-state concentrations of free radical species and repair oxidative cellular damage. Because ROS can cause damage

to virtually all cellular macromolecules, aerobic organisms have developed elaborate

antioxidant defense systems for the prevention of ROS-mediated cellular damage. ROS can be reduced enzymatically by antioxidant enzymes such as catalase, superoxide

dismutase (SOD), and glutathione peroxidase or nonenzymatically by antioxidants such

as vitamin E, vitamin C, and glutathione (Miquel, 2001). The antioxidant properties of green tea extract (Hong et al 2000), blueberry, spinach or strawberry (Joseph et al 1999),

Allium sativum (Pedraza- Chaverri et al 1998) and Glycyrrhiza glabra (Vaya et al 1997) have already proven beneficial in reducing oxidative tissue injury. Previous studies

111 suggest that the antioxidant vitamin E may inhibit some aspects of the aging process by

preventing lipid peroxidation. It is possible other antioxidants may have similar activity.

5.2 Oxidative stress and neurodengerative diseases

For years researchers have known that free radicals can cause cell degeneration, especially in the brain. Growing data from experimental models and human brain studies suggest oxidative stress may play an important role in neuronal degeneration in diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. The brain is particularly vulnerable to oxidative processes because it has a high glucose- driven metabolic rate, has low levels of antioxidant defense enzymes, contains high concentrations of polyunsaturated fatty acids, which are potential substrates for lipid peroxidation and is rich in enzymatically active transition metals, which can potentially catalyze radical formation (Halliwell and Gutteridge, 1989). High-dose α-tocopherol supplementation (5 g/kg) was associated with increased median and maximum life span

and improved brain mitochondrial function in male mice from a short-lived strain

(Navarro et al., 2005). Several studies have suggested that these increases in oxidative

stress vulnerability and the resulting neuronal loss can be reduced through dietary

supplementation of plant extracts that prevent brain atrophy as well as learning and

memory impairments (Kanowski et al 1996; Moriguchi et al 1997; Nishiyama et al

1997). It has been demonstrated that certain phenolic antioxidants attenuate neuronal cell death induced by oxidative stress (Schroeter et al 2000; Youdim and Joseph 2001).

Oxidative stress has been associated with both necrosis and apoptosis. In necrosis, a selective loss of membrane permeability occurs, which results in swelling of organelles,

112 loss of membrane depolarization, and rupture of the plasma membrane. Apoptosis is a

form of cell death that occurs during normal development and in pathological situations.

Cells undergoing apoptosis exhibit certain characteristics, including cytoplasmic

shrinkage, nuclear blebbing, and chromatin condensation (Kerr et al., 1972). Removal of

NGF from sympathetic neurons in vitro triggers a classic apoptotic death (Chang et al.,

2003) but progression along this cell death pathway can be aborted by readdition of NGF

to an NGF-deprived sympathetic neuron before a cell has reached the commitment-to-die

(Deckwerth and Johnson, 1993; Edwards and Tolkovsky, 1994).

Alzheimer's disease (AD) is the most common cause of dementia. This most

common of the late-life dementias is rising in prevalence with the aging of populations in developed countries and may now affect 20 to 30 million people worldwide (Selkoe,

2005). AD effects the hippocampus, cortex and amaygdala and is characterised by loss

memory loss and cognitive dysfunction. In people with Alzheimer's disease, deposits

called amyloid plaques build up in the brain. These are composed, in part, of a protein

called beta-amyloid, which is a fragment of the amyloid precursor protein (APP). A

mutation in this gene in humans is believed to be responsible for 5 to 20 percent of all

early onset familial Alzheimer's disease (Selkoe, 2005). Another neuropathological

characteristic of Alzheimer's disease are abundant filamentous tau lesions, intracellular

accumulations of abnormal filaments formed by the microtubule-associated protein tau.

Both amyloid plaques and neurofibrillary tangles (NFT) are clearly visible by microscopy

in brains of those afflicted by AD. Plaques are dense, mostly insoluble deposits of

amyloid-beta protein and cellular material outside and around neurons. They continue to

grow into insoluble twisted fibres within the nerve cell, often called tangles. Although

113 many older individuals develop some plaques and tangles as a consequence of aging, the

brains of AD patients have a greater number of them in specific brain regions such as the

temporal lobe. In vitro experiments support the observation that the neurotoxic effect of

Aβ is mediated by free radical mechanisms. Aβ aggregation produces oxidative stress in

neuronal cells and antioxidants such as vitamin E and melatonin attenuate this stress

(Behl et al. 1992). Vitamin E clearly had a beneficial effect in an Alzheimer's disease

clinical trial (Sano et al., 1997). The sympathetic nervous system is part of the autonomic

nervous system, which is responsible for maintaining homeostasis. Postganglionic

sympathetic neurons innervate endocrine and exocrine glands, cardiac muscle, and

smooth muscle throughout the body carrying out vasomotor, pilomotor, secretomotor,

sudomotor, and pupilodilator functions. The sympathetic nervous system regulates many

physiological processes, including body temperature, blood pressure, respiration, cardiac

output, blood glucose levels and gastrointestinal peristalsis (Glebova and Ginty, 2005). In response to an external or internal environmental change such as stress or a change in body posture, activation of the sympathetic nervous system affects multiple organ systems. Although an animal can survive without a sympathetic nervous system, it can do so only in conditions of constant ambient temperature and in the absence of stress. The importance of normal sympathetic function is underscored by the pathological states associated with sympathetic dysfunction, such as hypertension, congestive heart failure, dysautonomia, various neuropathies, and other disorders (De Quattro and Feng, 2002,

Goldstein et al., 2002, Low, 2002). Sympathetic neurons in culture die by apoptosis when

deprived of nerve growth factor (NGF). The neurotrophic factor hypothesis (Mattson,

1998) suggests that neurotrophic factor deprivation leads to formation of reactive oxygen

114 species (ROS) and a change in oxidant state (Dugan et al., 1997). The formation of free

radicals acts directly to destroy phospholipids and as signaling molecules to upregulate cell death pathways leading to apoptosis or necrosis (Greenlund et al., 1995). The increase of mitochondrial-derived reactive oxygen species (ROS) that occurs in these

NGF-deprived neurons has been demonstrated (Kirkland and Franklin, 2003). The prototypical model for investigating the cellular and molecular mechanisms underlying

this death consists of rodent sympathetic neurons dissociated from late embryonic or early

postnatal mice and grown in cell culture. Sympathetic neurons of this age require the

neurotrophin nerve growth factor (NGF) to live.

Figure 30. Morphological changes during the apoptotic death of sympathetic neurons deprived of NGF.

115 Depriving these cells of NGF causes their apoptotic death both in vivo and in vitro

(Kirkland et al., 2007; Figure 30). Thus, developing nerve growth factor (NGF)-

dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. During development of the rat, insufficient supply of NGF leads to the apoptotic death of approximately 40% of the sympathetic neurons in the superior cervical ganglion during the first few days after the animal is born (Wright et al., 1983). In a study done by Greenlund and co-workers (1995) microinjections of copper/ zinc SOD protein or a SOD expression vector delayed the apoptosis of sympathetic neurons while injection of an antisense SOD expression vector enhanced the death of neurons deprived of NGF.

In addition, using a redox-sensitive dye to analyze the production of ROS after NGF deprivation, sympathetic neurons displayed a peak in fluorescence intensity 3 hr after deprivation. These data indicate that in sympathetic neurons ROS are important mediators of apoptosis induced by NGF deprivation (Greenlund et al., 1995).

5.3 Pseudopterosins promote survival in NGF deprived mouse sympathetic neurons

The pseudopterosins have displayed potent antioxidant activity in both solution based and cell based assays (as described in previous chapters). With these results in mind we have attempted to test whether pseudopterosins promote the survival of postganglionic sympathetic neurons by reducing the oxidative burden. The survival of

these neurons was assessed in the absence of NGF, but in the presence of the compounds at varying concentrations. NGF was eliminated from the cultures with an antibody that

neutralizes the activity of NGF. Cells that were not deprived of NGF serve as a positive

116 the positivecontrol. 40 µMtreatments ofpsK2’acetateand4’ higher survivaloftheNGF deprivedsympat HSD (p<0.01)allthreeofthe 0.05; Appendix;Table15).Basedonthepa significantly differentacrossthetreatments analysisof variance (p< basedonaone-way the averageofviablecellsc control. Each treatment wasapplied tocellsintwowells.Thedatapresented represents w control. CellsdeprivedofNGFand HSD. deviation. Differentlettersindicat enhanced thesurvivalofNGF-deprivedmous Figure 31.

% survival versus +NGF 100.00 120.00 20.00 40.00 60.00 80.00 0.00 Treatment with40µMpseudopterosin

ps K 10 uM a

ps K 20 uM a ounted inthetwowells±standa ps K 40 uM b pseudopterosins testedat40µM

e significantlydifferentmeans (P ps K 2' ac 10 uM a ithout compound addedserveasthenegative

117 ps K 2' ac 20 uM a irwise comparison oftreatments byTukey’s acetate werenot significantly differentfrom hetic neurons(Figure e sympatheticneuron,N=2wells±standard

1 ps K 2' ac 40 uM c K, K2’ac,and4’acsignificantly

ps K 4' ac 10 uM a rd deviation.Themeans were ps K 4' ac 20 uM a resultedinsignificantly

31). Additionally,the ps K 4' ac 40 uM b,c

<0.01), Tukey’s NGF- a

NGF+ c

5.4 Pseudopterosins suppress ROS formation in NGF deprived sympathetic neurons

NGF withdrawal induces apoptosis in sympathetic neurons by causing the pro-

apoptotic protein, Bax, to bind to the outer mitochondrial membrane where it induces

release of cytochrome c from the mitochondrial intermembrane space into the cytoplasm

(Putcha et al., 1999). Increased levels of reactive oxygen species (ROS) occur in these

neurons as a result. The intensity of the fluorescent redox-sensitive dye MitoSox Red increases in NGF-deprived sympathetic neurons indicating that it has become oxidized by

the elevated levels of ROS in those cells (Kirkland et al., 2004). MitoSOX Red mitochondrial superoxide indicator is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells. MitoSOX Red reagent is live- cell permeant and is rapidly and selectively targeted to the mitochondria. Once in the mitochondria, MitoSOX Red reagent is oxidized by superoxide and exhibits bright red fluorescence upon binding to nucleic acids (Haugland, 2005). MitoSOX can also be oxidized by ROS other than superoxide but is most sensitive to oxidation by superoxide

(Kirkland et al., 2004). Pseudopterosins K and K4’ acetate were screened in vitro for the

ability to suppress ROS formation in sympathetic neurons deprived of NGF. Cultures

were deprived of NGF and stained with the superoxide-sensitive dye MitoSOX 24 hr

later. Deprived cultures were treated with various concentrations of the compounds at the time of deprivation.

Dye intensity was measured only in the cytoplasm. Nuclear staining occurred only

in a small proportion of apoptotic cells that appeared to have compromised plasma

membranes. Neurons in which cytoplasm could not be clearly differentiated from the

nucleus were excluded from analysis. MitoSOX was excited with the 408 nm line of the

118 confocal laser Tukey’s HSD. with pseudopterosins.Diffe Figure 32. NGF-deprived cells(Tukey’s HSDp<0.01,Figure32,33). 10 µMtreatment Kshowedasignificantreduction in ROSversusthe ofpseudopterosin means differedsignificantlyacrosstreatme neurons.A one-w in NGF-deprivedsympathetic concentration ofdyeforthesame maintained The measured intensity for image acquisition.Generally,eightseparate

% fluorescence vs NGF+ control 100 150 200 250 300 350 400 450 50 0 The pseudopterosinstestedgreatlysuppr QuantificationofROSinNGFdepriv

in thestandard culturemedium

and theredphotomultiplier channel

NGF+ a,c

of eachneuronwasnormalized to K 10 uM b rent lettersindicatesigni periodasdidtheexperimental K 20 uM a 119 nts (p<0.05,Appendix;Table16).Allbutthe K 40 uM a containing NGFandreceivingthe containing essed theincreaseof

fields ofviewwerescannedperplate. ay analysisofvarianceconfirmed that ed mouse sympathetic neuronstreated

of theconfocalmicroscope wasused K 4' ac 10 uM a ficantly different means ( means different ficantly

K 4' ac 20 uM a thatofcontrol neurons

cells.

MitoSOXintensity K 4' ac 40 uM a,c

NGF- b P <0.01),

same

b a

c d

Figure 33. Confocal micrographs of sympathetic neurons exposed to the dye MitoSox Red a) NGF- (negative control) b) NGF + (positive control) c) ps K 40µM d) ps K 4’ acetate 40µM

120 Many neurodegenerative disorders and syndromes are associated with an

excessive generation of reactive oxygen species (ROS) and oxidative stress. Direct

antioxidants such as chainbreaking free radical scavengers can prevent oxidative nerve

cell death. Although there is ample experimental evidence demonstrating neuroprotective activities of direct antioxidants in vitro, the clinical evidence for antioxidant compounds

to act as protective drugs is relatively scarce. Recent research efforts into the elucidation

of the mechanisms of neurodegeneration in Alzheimer’s disease (AD), Parkinson’s

disease (PD), stroke, and other neurodegenerative disorders has led to an increased

understanding of the basic mechanisms of nerve cell death. In general, cell death can follow two basic pathways. Apoptosis (also called programmed cell death) is an active form of cell degeneration and is executed by enzymes (caspases). Necrosis, the other type of cell death, is frequently occuring during acute insults and is characterized by rapid cell lysis. Both processes of cellular degeneration are frequently accompanied by an overshooting generation of free radicals and other cellular events leading to oxidative stress (Behl and Moosmann, 2002). This final oxidative pathway of nerve cell death could be a basis for preventive and therapeutic avenues. With respect to neurodegenerative diseases and syndromes, multiple pathogenetic mechanisms may be involved upstream, ranging from inflammation to disturbed energy metabolism, many of which are accompanied by oxidative stress (Behl and Moosmann, 2002). Antioxidants in a pharmacological sense are compounds which counteract the adverse effects of oxidative stress. They can be differentiated with respect to their modes of action, which include the inhibition of free radical formation (indirect antioxidants), the direct chemical scavenging of generated free radicals (direct antioxidants), and/or the strengthening of the cellular

121 capability to cope with high ROS loads via enzymatic processes which detoxify accumulating ROS, or to promote repair of the effected damage (Behl and Moosmann,

2002).

Based on the preliminary results presented here, pseudopterosins might be a promising drug candidate in the treatment of those neurodegenerative diseases in which oxidative stress plays an important role. Convincing evidence exist showing that different classes of antioxidants, mainly those that belong to the family of direct antioxidants, act as neuroprotectants in vitro. Vitamin E remains to be one of the most potent chain- breaking antioxidant structures. However, its passage through the blood brain barrier

(BBB) is limited. Consequently, the identification of structures with much better BBB penetration and increased antioxidant activity may, ultimately, lead to the development of novel neuroprotective drugs.

122 CHAPTER 6

Methods and materials

Collection and identification of coral material. Fragments of Pseudopterogorgia sps colonies were collected by SCUBA at a range depth of 5–30 m at various sites around the the Bahamas and Key Largo. Sample collection never implied removing whole colonies, only a terminal fragment of each individual colony was cut off the main gorgonian axis with sharp scissors. Gorgonian fragments were air-dried and stored in the freezer.

Samples were kept frozen until the moment of extraction. Taxaonomic identification of each colony was made by comparison of the gross colony architecture and by morphology and dimensions of the three most common sclerite types (i.e., spindles, anthocodial rods, and scaphoids). To obtain sclerites, we clipped off a small fragment of the colony and digested it in household bleach. Once the organic tissue was digested, sclerites were repeatedly washed with distilled water and ethanol, and dried in an oven at

70±C for 24 hr. Crude sclerite preparations were mounted on glass slides and analyzed under a microscope. For each colony, we measured 30 anthocodial rods, 30 spindles, and

30 scaphoids. For species designations, we followed Bayer (1961).

Solid-Phase Extraction The organic extracts are fractionated using a solid phase

Extraction (SPE) 12-port vacuum manifold. The organic extract (5 - 15 mL) is concentrated on to polymeric HP20 (3 g) using a savant vacuum centrifuge system. The

123 HP20 is transferred into a 40-mL syringe-barrel SPE column, after washing of the

column with water (20 mL) the column is eluted drop-wise with 15 mL fractions of; 1)

40% acetone/water 2) 75% acetone/water, and 3) 100% acetone. The eluent is collected

in scintillation vials (20 mL) and is dried in a vacuum centrifuge.

Semi-preparative HPLC separation. HPLC separations were performed on a

Shimadzu HPLC system consisting of a Shimadzu LC-20AT quaternary solvent delivery

system, SPD-M20A Photodiode array detector, evaporative light scattering detector

(ELSD). We are using an evaporative light scattering detector to estimate the quantity of

compounds present in the fraction. A sample of the 75% acetone/water fraction (5 mg) is

subjected to semi-preparative HPLC separation on reversed phase. A post-column fixed

flow splitter is used to split the flow in a ratio of 1:20 to the ELSD and the fraction

collector, respectively. The elution protocol for the HPLC separation is isocratic elution

with 20% acetonitrile in water for 2.5 min, followed by a linear gradient of Acetonitrile

from 20-90 % in 35 min, followed by isocratic elution with 90% for 10.0 min.

Collection of larvae. Pseudopterogorgia elisabethae were collected from spawning

adult colonies in November 2004 at the marine aquaculture facility at the Gumbo Limbo

Environmental Center in Boca Raton, Florida. These adult colonies had been collected by

SCUBA at a depth of 15 M from Long Key, Florida in October, 2004. The larvae were

then transferred to 1 L glass containers with Millepore (0.22µm) filtered seawater (~200 larvae/jar). Pseudopterogorgia bipinnata colonies were collected at Long Key, Fl by

SCUBA at a depth of 25m in December of 2004. Spawning adult colonies were collected

124 and placed in individual sealed bags of seawater. Once transported to our lab in Boca

Raton, larvae were separated from adult colonies by pipette, rinsed 3X in MFSW and

placed in 1L glass containers of MFSW. All larval cultures were maintained in a growth

incubator at 26˚ C with a 12/12 light/dark cycle and 40% of the water was changed once

every second day while larvae were in culture.

3 Incubation of gorgonian larvae with [C1- H] geranylgeranyl diphosphate. For each

species, three replicates of 100 larvae each were transferred to 125 mL glass jars

3 containing 100 mL of MFSW and were incubated with 4µCi of C1- H-GGDP (50-60

Ci/mmol) at 26ºC and 200RPM on an environmental shaker for 24 h. Larvae were then

transferred in a minimal amount of seawater (~1 ml) into 15 mL conical tubes. 9 mL of

MeOH was added, and the tubes were centrifuged.

Extraction and purification of diterpenes from larvae. Larvae were extracted in

MeOH/H2O (9:1) (at room temperature for ~ 1hr) and partitioned with hexanes. The

aqueous partitions were then adjusted to MeOH/ H2O (1:1), and partitioned a second time

with CH2Cl2. Normal phase HPLC purification was performed using a Vydac semi preparative column. For isolation of diterpenes from P.bipinnata larval CH2Cl2 extract, a

mobile phase gradient from 100% hexanes to hexanes/EtOAc (50:50) with UV detection

at 270nm. Reverse phase HPLC was performed using a semi preparative Phenomenex

phenyl-hexyl column and a mobile phase gradient of MeOH/H2O (50:50) to MeOH

(100%) with UV detection at 270nm. HPLC conditions for isolation of pseudopterosins from P.elisabethae larval CH2Cl2 extract used a mobile phase gradient from

125 hexanes/EtOAc (60:40) to 100% ethyl acetate with detection at 283 nm. Pseudopterosin

C was further purified by reversed phase HPLC using a gradient of acetonitrile/water (50-

100%) as mobile phase and detection at 283nm. 1H-NMR spectra were recorded at 400

MHz in CDCl3 on a Varian Innova spectrometer.

Thin layer chromatography. TLC was carried out on pre-coated silica gel plates 60 F254 or RP-18 F254 plates with 0.5 or 1 mm film thickness (Whatman) with a mobile phase of

Hexanes/EtOAc (60:40). Spots were visualized by UV light followed by spraying the plate with 10% H2SO4 in MeOH and charring with a heat gun.

Zooxanthellae Isolation. Coral tissue was homogenized in a Waring blender using

deionized water. The resulting slurry was divided into 50mL Falcon tubes and

centrifuged at 350 x g for 5 min. The supernatant was decanted and the pellet

resuspended in deionized water. This was repeated until the supernatant was clear. The

pellet was resuspended, layered over 100% Percoll, and centrifuged at 38 x g to pellet

spicules. The zooxanthellae formed a brown band above the Percoll, which was

transferred to another Falcon tube using a plastic pipet. This cell layer was diluted with

deionized water and layered over a discontinuous gradient of 30, 70, and 100% Percoll

and centrifuged at 350 x g for 30 minutes. The layer of cells was cleaned three additional

times in the same manner until no further debris was observed under a light microscope.

PCR analysis of DNA from isolated zooxanthellae and larvae. All DNA was extracted

using UltraClean™ Soil DNA Kit (MoBio Laboratories, inc) according to the

126 manufacturer’s protocol. The isolated DNA was quantified and assessed for purity by measuring the absorbance at 260 and 280 nm and calculating the A260:A280 ratio. The

DNA was aliquoted at 50 ng μl–1 and stored at –80°C until used. The internal transcribed spacer region (ITS-rDNA) was PCR-amplified using the zooxanthellae- specific primers designed to amplify between the conserved regions of the 3’ and 5’ ends of the zooxanthellae 18S and 28S rDNA genes (Santos et al. 2001). Octocoral DNA was amplified using primers designed to target microsatellite loci in P elisabethae reported by

Gutierrez-Rodriguez and Lasker (2004). In general, 1 μl of DNA (50 ng μl–1) was used in a 50 μl PCR reaction containing 0.5 μl Taq (2.5 U; Qiagen), 5 μl 10× buffer, 3 μl 25 mM MgCl2, 1 μl 100 mM dNTPs, 1 μl of each primer (10mM concentration). PCR

cycling conditions were an initial denaturing period of 2 min at 94°C followed by 30

cycles of 94°C for 40 s, 53°C for 1 min, and 72°C for 1 min. A final extension of 30 min

at 72°C was added. 5 to 10 % of the reaction was loaded on a 1 % agarose gel (0.6%

Metaphor agarose/0.6% Seakem agarose; Fisher Scientific), run at 90 V, and visualized by ethidium bromide.

Antibiotic/antimycotic treatment of larvae. 3 containers of 100 ml MFSW with ~100 larvae were treated with antibiotic/antimycotic twice over 48 hours with final concentrations as follows: 0.1 mg/ml kanamycin (a water-soluble, broad-spectrum antibiotic), 0.1 mg/ml streptomycin, 100 units/ml penicillin, 0.25 g/ml amphotericin B.

At the end of the 48 hours the samples were incubated for 24 hrs with the tritiated diterpene precurser and then extracted and analysed as previously described.

127 Acid hydrolysis of pseudopterosins A-D to pseudopterosin aglycone. To a mixture of

pseudopterosins A-D was added a 1N HCl methanolic solution (2 mL). The reaction vessel was agitated at 50oC for 3 hours and then the solution was cooled to 25oC, and

water added (10 mL). The solution was extracted with methylene chloride (3 x 2 mL),

the organic extracts combined, dried over anhydrous sodium sulfate, and the solvent

allowed to evaporate under a nitrogen. The residue was purified by semi-preparative RP-

C18 HPLC (λ = 283 nm) using a linear gradient of acetonitrile:water (50:50

acetonitrile:water to 100% acetonitrile over 30 minutes, hold for 10 minutes) as mobile

phase.

Culture of rat sympathetic neurons. Superior cervical ganglia were dissected from

Sprague–Dawley rat fetuses (Harlan Bioproducts) on embryonic day 20 or 21. Neurons

were enzymatically and mechanically dissociated from the ganglia and maintained in cell

culture. Culture medium consisted of Eagle’s minimum essential medium with Earle’s

salts (Life Technologies, Inc., Gaithersburg, MD, U.S.A.) supplemented with 10% fetal

bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 μM fluorodeoxyuridine,

20 μM uridine, 1.4 mM L-glutamine, and 50 ng/ml 2.5S NGF. Cells used in survival

assays were plated on a collagen substrate in 24-well Costar tissue culture dishes

(Corning, Inc., Corning, NY, U.S.A.). Those used for fluorescent or confocal microscopy

were plated on a collagen substrate coated on glass coverslips glued over holes cut in the

bottoms of 35-mm Falcon tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ,

U.S.A.). One-half to one ganglion was plated per culture in all experiments. NGF was

withdrawn from cells by incubating cultures in the culture medium containing a NGF-

128 neutralizing antibody (Harlan Bioproducts) and lacking NGF. Cells had been in culture

for 6–9 days at the beginning of experiments.

Quantification of ROS in sympathetic neurons. MitoSOX Red was purchased from

Invitrogen (Eugene, OR). Cultures were incubated for 10 min at 35°C in the appropriate

experimental medium containing MitoSOX (2 µM). A Nikon (Melville, NY) C1 laser-

scanning confocal microscope mounted on a Nikon Eclipse TE 300 inverted microscope

was used for all confocal microscopy. The confocal microscope was controlled by EZC1

software running on a Dell computer. Neurons, observed with a 60x plan oil immersion

lens (numerical aperture, 1.4), were chosen at random and scanned by the confocal

microscope. Laser power, confocal pinhole size, and photomultiplier gain were maintained at constant levels during an experiment. After incubation in MitoSOX, cultures were washed twice with L-15 medium and kept in the second wash for

microscopy. MitoSOX was excited with the 408 nm line of the confocal laser and the red

photomultiplier channel of the confocal microscope was used for image acquisition.

FRAP assays. FRAP reagent was prepared before each assay with 300 mM acetate

buffer, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution, and 20 mM FeCl3 in a 10:1:1 ratio. Reagent was heated to 37° prior to use. Assays were prepared in 96-well microtiter plates using 150 μL of warm FRAP reagent with 20 μL of sample. A blank reading of the plate was taken before addition of samples. Following addition of the sample compounds,

absorbance readings at 593 nm were recorded at 30, 90, and 120 minutes (SpectraMax

129 M2 microplate reader; Molecular Devices). The 90 min reading was selected for calculation of FRAP equivalence values. Standard curve were produced using 50, 125,

250, 500, and 1000 µM of FeSO4 and 5, 10, 20, 50 and 100 µM of Trolox.

DCFH-DA assays. DCFH-DA (Sigma-Aldrich) was prepared as a 100 mM stock solution in MeOH and stored away from light at -20°C. Cells were centrifuged briefly to remove media, and incubated in 10 μM DCFA-DA probe for 30 min with constant agitation. Following pre-loading with the probe, the probe was removed and cells resuspended in 36 mL seawater. A total of 190 μL cell suspension was added to each well of black-bottom 96-well microtiter plates. Cells were maintained in a water bath at 32°C and subjected to radiation stress under a UV lamp for 3 h. Fluorescent intensity was recorded using a monochromatic spectrophotometer (SpectraMax M2 microplate reader;

Molecular Devices) set at excitation wavelength 485 and emission wavelength 538.

Photoinhibition assay. Cultured Symbiodinium sp (Clade B2) came from the lab of Dr

G. Schmidt, Department of Plant Biology, University of Georgia. 1 ml of ~ 106 cells/ml zooxanthellae were transferred to a flat bottom 1.2 ml vial. The samples was dark acclimated at rt for twenty minutes prior to initial PAM reading (PAM measurement was recorded using a Diving-PAM, Walz, Germany). The vial was maintained at 34 ºC using a water bath and exposed to light at ~300 µM for 35 minutes, followed by 5 minutes of dark and a PAM reading. This cycle was repeated 14 times. All compounds tested were dissolved in 10 µL DMSO, and DMSO only controls contained 10 µL DMSO.

130 Statistical analysis. In most cases, analysis of variance followed by nonparametric

Tukey multiple comparison test was performed using the GraphPad Prism 5 (GraphPad

Software, San Diego, CA, USA).

Purification and structure elucidation. Optical rotation was measured on a Bellington

Stanley Ltd. ADP-220 digital polarimeter. UV spectra were obtained on a Perkin-Elmer

-15 UV spectrophotometer. IR spectra were recorded on a Thermo Electronic

Corporation Nicolet IR-100 spectrophotometer. All 1D and 2D NMR spectra were performed on a Varian Inova 500 MHz NMR spectrometer, and chemical shifts are expressed in parts per million (δ) relative to tetramethylsilane as internal reference.

HPLC purifications were performed on Beckman Coulter 126P solvent module system using preparative HPLC column (Phenomenex Gemini 5µ, C18, 110A, 250 x 21.2 mm).

The structures of the new compounds were established using chemical and spectroscopic

(ESIMS, 1H NMR, 13C NMR, COSY, HSQC, HMBC, ROESY) studies, whereas the known compounds were identified by comparison of their spectroscopic data (UV, MS and NMR) with published reports.

Instrumentation. Solid phase Extractions (SPE) were performed on HP20 resin (3.0 g) in a 40-mL syringe-barrel column using a 12-port manifold (Altech Associates Inc).

Semi-preparative HPLC separation were performed on a PRP-1 column (10 x 250 mm,

10 µm, Hamilton) using a Shimadzu HPLC system consisting of a Shimadzu DGU-20A online degasser, Shimadzu LC-20AT quaternary solvent delivery system, SPD-M20A

Photodiode array detector, a Shimadzu LTII evaporative light scattering detector (ELSD)

131 and a FRC-10A fraction collector (a QuickSplit™ Flow Splitter (ASI, El Sobrante, CA)

was used to split the flow in 1:20 to the ELSD and fraction collector). The system was

controlled with an SCL-10AVP system controller using EZStart chromatography

software. Liquid handling is performed using a Precision XS microplate sample processor

(BioTek Instruments, Inc, Winooksi, VT).

25 Pseudopterosin K 2’ acetate: amorphous solid; []D – 117.6 (c 0.017, CHCl3); UV (MeOH) max 228 (ε 31232), 277 (ε 5477), 284 (ε 6075) nm; IR (film) νmax 3344, 2927, -1 1 13 1745, 1456, 1432, 1374, 1242 cm ; H (CDCl3, 500 MHz) and C (CDCl3, 125 MHz) NMR spectra, see Table 1; HRESIMS (positive-ion) m/z [M + Na]+ 511.2655 (calcd for C28H40O7Na, 511.26717). 25 Pseudopterosin K 4’ acetate: amorphous solid; []D – 130.4 (c 0.046, CHCl3); UV (MeOH) max 228 (ε 36892), 277 (ε 6197), 284 (ε 6871) nm; IR (film) νmax 3395, 2925, -1 1 13 1732, 1431, 1375, 1241 cm ; H (CDCl3, 500 MHz) and C (CDCl3, 125 MHz) NMR spectra, see Table 1; ESIMS (positive-ion) m/z 489 [M + H]+; HRESIMS (positive-ion) + m/z [M + H2O] 506.3163 (calcd for C28H42O8, 506.2880). 25 Pseudopterosin Iso-A: amorphous solid; []D – 130.4 (c 0.046, CHCl3); UV (MeOH) max 228 (ε 15471), 277 (ε 3413), 284 (ε 3309) nm; IR (film) νmax 3365, 2925, 1457, -1 1 13 1312, 1248 cm ; H (CDCl3, 500 MHz) and C (CDCl3, 125 MHz) NMR spectra, see + Table 2; HRESIMS (positive-ion) m/z [M + Na] 455.2415 (calcd for C25H36O6Na, 455.2409). 25 Pseudopterosin Iso-C: amorphous solid; []D – 130.4 (c 0.05, CHCl3); UV (MeOH) max 228 (ε 16003), 277 (ε 4266), 284 (ε 3555) nm; IR (film) νmax 3394, 2926, 1732, -1 1 13 1456, 1374, 1236 cm ; H (CDCl3, 500 MHz) and C (CDCl3, 125 MHz) NMR spectra, + see Table 2; ESIMS (positive-ion) m/z 492 [M + H2O] ; HRESIMS (positive-ion) m/z [M + + H] 475.2693 (calcd for C27H39O7, 475.2695). 25 Pseudopterosin Iso-D : amorphous solid; []D – 133.3 (c 0.015, CHCl3); UV (MeOH) max 228 (ε 15879), 277 (ε 3126), 284 (ε 3056) nm; IR (film) νmax 3394, 2926, 1741, -1 1 13 1456, 1373, 1235 cm ; H (CDCl3, 500 MHz) and C (CDCl3, 125 MHz) NMR spectra, + see Table 2; HRESIMS (positive-ion) m/z [M + H] 475.2693 (calcd for C27H39O7, 475.2695).

132 REFERENCES

Abramson, S.N., Trischman, J.A., Tapiolas, D.M., Harold, E.E., Fenical, W., Taylor, P. 1991. Structure / activity and molecular modeling studies of the lophotoxin family or irreversible nicotinic receptor antagonist. J. Med. Chem. 34:1798-1804.

Agrawal, A.A., Karban, R. 2000. Specificity of constitutive and induced resistance: pigment glands influence mites and caterpillars on cotton plants. Entomol. Exp. Appl. 96:39

Agrawal, A.A. 2001. Phenotypic plasticity in the interactions and evolution of species. Science 294:321−326.

Aksenov, M. Y., Butterfield, D. A., Geddes, J. W., Markesbery, W. R. 2001. Protein oxidation in the brain in Alzheimer’s disease. Neuroscience. 103:373–383.

Andermann, A.J. 1996. Physicians, Fads, and Pharmaceuticals: A History of Aspirin. M.J.M. 2:115-120.

Anggadiredja, J., Andyani, R., Hayati, Muawanah. 1997. Antioxidant activity of Sargassum polycystum (Phaeophyta) and Laurencia obtusa (Rhodophyta) from Seribu Islands. J. App. Phycol. 9:477–479.

Anthoni, U., Nielsen, P.H., Pereira, M., Christophersen, C. 1990. Bryozoan secondary metabolites: a chemotaxonomical challenge. Comp Biochem Physiol B. 96:431–437.

Arai, T., Kato, M., Heyward, A. 1993. Lipid-composition of positively buoyant eggs of reef building corals. Coral Reefs, 12: 71–75.

Arif, J.M., Al-Hazzani, A.A., Kunhi, M., Al-Khodairy, F. 2004. Novel marine compounds: Anticancer or Genotoxic. J. Biomed. Biotech. 2:93-98.

Aruoma, O.I., Halliwell, B., Aeschbach, R., Löliger, J. 1992. Antioxidant and pro-oxidant properties of active rosemary constituents: carnosol and carnosic acid. Xenobiotica 22:257-268.

Bandurraga, M. M., Fenical, W., Donovan, S. F., Clardy, J. 1982. Pseudopterolide, an irregular diterpenoid with unusual cytotoxic properties from the Caribbean sea whip Pseudopterogorgia acerosa (Pallas) (Gorgonacea). J. Am. Chem. Soc. 104:6463-6465.

133 Bandurraga, M.M., Fenical, W. Isolation of the muricins. 1984. Evidence of a chemical adaptation against fouling in the marine octocoral Muricea fruticosa (Gorgonacea). Tetrahedron. 41:1057-1065.

Banin, E., Vassilakos, D., Orr, E., Martinez, R. J. & Rosenberg, E. 2003. Superoxide dismutase is a virulence factor produced by the coral bleaching pathogen Vibrio shiloi. Curr. Microbiol. 46:418–422.

Baskin, Y. 1997. The Work of Nature: How the Diversity of Life Sustains Us. Island Press, New York.

Bayer, F.M. The shallow-water Octocorallia of the West Indian region. A manual for marine biologists. 1961, Martinus Nijhoff, The Hague.

Beal, M. F. 1995. Aging, energy and oxidative stress in neurodegenerative diseases. Ann. Neurol. 38:357–366

Beal, M. F. 1997. Oxidative damage in neurodegenerative diseases. Neuroscientist 3:21–27

Beal, M.F. 2002. Oxidatively modified proteins in aging and disease. Free Radic. Biol. Med. 32:797–803.

Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A. 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad Sci. 87:1620-24.

Behl, C., Moosmsnn, R. 2002. Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds. Biol. Chem. 383:521-536.

Benzi, G., Moretti, A. 1995. Are reactive oxygen species involved in Alzheimer’s disease? Neurobiol. Aging 16:661–674.

Benzie, I.F.F., Strain, J.J. 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration: Methods Enzymol. 299:15–34.

Benzie, I.F.F., Stezo, Y.T. 1999. Total antioxidant capacity of teas by the ferric reducing/antioxidant power assay. J. Ag. Food Chem. 47:633–636.

134 Benzie, I.F.F., Strain, J.J. 1996. The ferric reducing ability of plasma as a measure of “antioxidant power” the FRAP assay. Anal. Biochem. 239:70–76.

Bergmann, W., Creighton, S.M., Stokes, W.M. 1956. Contributions to the study of marine products. XL. Waxes and triglycerides of sea anemones. Journal of Organic Chemistry 21:721–728.

Berkelmans, R., Oliver, J.K.1999. Large scale bleaching of corals on the Great Barrier Reef. Coral Reefs 18:55–60.

Berkelmans, R., van Oppen, M.J.H. 2006. The role of zooxanthellae in the thermal tolerance of corals: a ‘nugget of hope’ for coral reefs in an era of climate change. Proc. R. Soc. Lond. B. Biol. Sci. 273:2305–2312.

Bhagooli, R., Hidaka, M. 2004. Release of zooxanthellae with intact photosynthetic activity by the coral Galaxea fascicularis in response to high temperature stress. Marine Biology 145:329-337.

Bindokas, V. P., Jordan, J., Lee, C. C., Miller, R. J. 1996. Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine. J. Neurosci. 16:1324–1336.

Birkeland, C. (ed.). 1997. Life and Death of Coral Reefs. Chapman and Hall, New York.

Björkman, O. and Demmig, B. 1987. Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170:489–504.

Blokhina, O., Virolainen, E., Fagerstedt, K.V. 2003. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Anal. Bot. 91:179–194.

Blunt, J.W., Copp, B.R.,Munro, M.H., Northcote, P.T., Prinsep, M.R. 2004. Marine Natural Products. Nat. Prod. Rep. 1: 1 – 49.

Boehnlein, J.M., Santiago-Vasquez, L.Z., Kerr, R.G. 2005. Diterpene biosynthesis by the dinoflagellate symbiont of the Caribbean gorgonian Pseudopterogorgia bipinnata. Mar. Ecol. Prog. Ser. 303:105-111.

Bradshaw, A.D. 1965. Evolutionary significance of phenotypic plasticity in plants. Adv. Genet. 13:115–155.

Brown, B. E., Downs, C. A., Dunne, R. P. and Gibb, S. W. 2002. Exploring the basis of thermotolerance in the reef coral Goniastrea aspera. Mar. Ecol. Prog. Ser. 242:119 -129.

Brown, B. E. 1997. Coral bleaching: causes and consequences. Coral Reefs 16:129-138.

135 Bruckner, A.W. 2002. Life-saving products from coral reefs. Issues Sci. Techn. 18:39– 44.

Bryant, D., L. Burke, J. McManus, Spalding, M. 1998. Reefs at risk: a map-based indicator of threats to the world's coral reefs. World Resources Institute, Washington, D.C.

Buckingham, J. 1998.Dictionary of Natural Products on CD-ROM, volume 6.1. London: Chapman & Hall.

Buddemeier, R. W., Fautin, D. G. 1993. Coral bleaching as an adaptive mechanism. BioScience 43:320-326.

Buddemeier, R.W., Smith, S.V. 1999. Coral adaptation and acclimatization: a most ingenious paradox. Am. Zool. 39:1–9.

Cadenas, E. 1989. Biochemistry of oxygen toxicity. Annu. Rev. Biochem. 58:79–110.

Carroll, C.R., Hoffman, C.A. 1980. Chemical feeding deterrent mobilized in response to insect herbivory and counteradaptation by Epilachna tredecimnotata. Science. 209:414– 416.

Carpenter, R.C. 1986. Partitioning herbivory and its effects on coral reef algal communities. Ecol. Monogr. 56: 345-365.

Carte, B. K. 1996. Biomedical potential of marine natural products. BioScience 46:271- 86.

Castanaro J., Lasker, H.R. 2003 Effects of clipping on growth of colonies of the Caribbean gorgonian Pseudopterogorgia elisabethae. Invertebr Biol 122:299–307.

Cetrulo, G. L. and Hay, M. E. 2000. Activated chemical defenses in tropical versus temperate seaweeds. Mar. Ecol. Prog. Ser. 207:243–253.

Chan, W. R., Tinto, W. F., Laydoo, R. S., Machand, P. S., Reynolds, W. F., McLean, S. 1991. Cembrane and pseudopterane diterpenoids of the octocoral Pseudopterogorgia acerosa. J. Org. Chem. 56:1773-1776.

Chang, L.K., Schmidt, R.E., Johnson, E.M. 2003. Alternating metabolic pathways NGF- deprived sympathetic neurons affect caspase-independent death. J. Cell Biol. 162: 245– 256.

Chanwitheesuk, A., Teerawutgulrag, A., Rakariyatham, N. 2005. Screening of antioxidant activity and antioxidant compounds of some edible plants of Thailand. Food Chem. 92:491–497.

136 Cheeseman, K.H., Slater, T.F. 1993. An introduction to free radical biochemistry. Br. Med. Bull. 49: 481–493.

Chew, F.S. 1988. Biological effects of glucosinolates. In: Cutler GG (ed) Biologically active natural products: potential use in agriculture. Am. Chem. Soc. Symp. Ser. 380:155–181.

Chew, Y.L., Lim, Y.Y., Omar, M., Khoo, K.S. 2008. Antioxidant activity of three edible seaweeds from two areas in South East Asia. Food Sci. Tech. 41:1067-1072.

Ciereszko, L.S., Sifford,D.H.,Weinheimer, A.J. 1960. Chemistry of coelenterates. Occurrence of terpenoid compounds in gorgonians. Annals of New York Academy of Sciences. 90: 917-919.

Ciereszko, L.S., Johnson, M.A., Schmidt, R.W., Koons, C.B. 1968. Chemistry of Coelenterates-VI. Ocurance of Gorgosterol, A C30 Sterol, in Coelenterates and their zooxanthellae. Comp. Biochem. Physiol. 24:899-904.

Ciereszko, L.S., Guillard ,R.R.L. 1989. The influence of some cembranolides from gorgonian corals on motility of marine dinoflagellates. J. Exp. Mar. Biol. Ecol.127:205- 210.

Clausen T., Reichardt, P., Bryant, J., Werner, R., Post, K., Frisby, K. 1989. Chemical model for short-term induction in quaking aspen (Populus tremuloides) foliage against herbivores. J. Chem. Ecol. 15: 2335–2346.

Climate Resource Board. 1979. Carbon Dioxide and Climate: A Scientific Assessment. Report of an Adhoc Study Group on Carbon Dioxide and Climate.

Coffroth, M., H. Lasker, and J. K. Oliver. 1990. Coral mortality outside of the eastern Pacific during 1982-83: relationship to El Niño. Pages 141-147 in P. Glynn (ed.). Global Consequences of the 1982-82 El Niño -Southern Oscillation. Elsevier Press, Amsterdam.

Conolly, J.D. and R.A. Hill. Dictionary of Terpenoids. Chapman and Hall: New York, 1992.

Cook, C., A. Logan, J. Ward, B. Luckhurst, and C. Berg. 1990. Elevated temperatures and bleaching on a high latitude coral reef: the 1988 Bermuda event. Coral Reefs 9:45- 49.

Coyle, J. T., Puttfarcken, P. 1993. Oxidative stress, glutamate and neurodegenerative disorders. Science 262:689–695.

Cragg, G.M., Newman, D.J. and Snader, K.M. 1997. Natural Products in Drug Discovery and Development. J. Nat. Prod. 60:52-60.

137 Cronin, G.,Hay M.E., Fenical W.,Lindquist, N. 1995. Distribution, density, and sequestration of host chemical defenses by the specialist Tritonia hamnerorum found at high densities on the sea fan Gorgonia ventalina. Mar. Ecol. Prog. Ser. 119:177-189.

D'Croz, L., Mate-T., J. L. 2004. Experimental responses to elevated water temperature in genotypes of the reef coral Pocillopora damicornis from upwelling and non-upwelling environments in Panama. Coral Reefs 23: 473-483.

Dalle-Donne, I., Scaloni, A., Giustarini, D., Cavarra, E., Tell, G., Lungarella, G., Colombo, R., Rossi, R., Milzani, A. 2005. Proteins as biomarkers of oxidative/nitrosative stress in diseases: The contribution of redox proteomics. Mass Spectrom. Rev. 24:55–99.

Davidson, S. K., Allen, S. W., Lim, G. E., Anderson, C. M., Haygood, M. G. 2001. Evidence for the biosynthesis of bryostatins by the bacterial symbiont “Candidatus Endobugula sertula” of the bryozoan Bugula neritina. Appl. Environ Micro. 67:4531– 4537.

Davies, M.J., Forni, L.G., Willson, R.L. 1988. Vitamin E analogue Trolox C: E.s.r. and pulse-radiolysis studies of free-radical reactions. Biochem J. 255: 513-522.

DeQuattro V, Feng M. 2002. The sympathetic nervous system: the muse of primary hypertension. J. Hum. Hypertens. 16: S64–69.

Deckwerth, T.L., and E.M. Johnson, Jr. 1993. Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J. Cell Biol. 123:1207–1222.

Denisov, E.T., Afanas’ev, I.B. 2005. Oxidation and antioxidants in organic chemistry and biology. Publisher: Boca Raton, FL : Taylor & Francis.

Done, T. J., Ogden, J. C., Wiebe, W. J. 1996. Biodiversity and ecosystem function of coral reefs. Pages 393-429 in Mooney, H. A., J. H. Cushman, E. Medina, O.E. Sala, and E.D. Schulze (eds.). Functional Roles of Biodiversity: A Global Perspective. John Wiley & Sons, Chichester, U.K.

Douglas, A. E., 2003. Coral bleaching--how and why? Mar Pollut Bull. 46(4):385-92.

Downs, C. A. , Fauth , J. E. , Halas, J. C., Dustan, P., Bemis, J., Woodley, C.M. Oxidative stress and seasonal coral bleaching. Free Radical Biology and Medicine. 2002, 33, 533-543.

Duval, B., Shetty, K., Thomas, W.H., 2000. Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light. J. App. Phycol. 11:559-566.

138 Dykens, J. A., Shick, J. M. 1984. Photobiology of the symbiotic sea anemone Anthopleura elegantissima: Defense against photo-dynamic effects, and seasonal photoacclimatization. Biol. Bull. 167:693-697.

Eakin, C. M. 1996. Where have all the carbonates gone? A model comparison of calcium carbonate budgets before and after the 1982-1983 El Niño. Coral Reefs 15:109-119. Edwards, S.N., A.M. Tolkovsky. 1994. Characterization of apoptosis in cultured rat sympathetic neurons after nerve growth factor withdrawal. J. Cell Biol. 124:537–546.

Elyakov G.B., Kuznetsova T., Mikhailov V.V., Maltsev I.I., Voinov V.G., Fedoreyev, S.A. 1991. Brominated diphenyl ethers from a marine bacterium associated with the sponge Dysidea sp. Experientia. Mar. Ecol. Prog. Ser. 47:632–633.

Epifano, R.D., Martins, D.L., Villaca, R., Gabriel, R. 1999. Chemical defenses against fish predation in three Brazilian octocorals: 11 beta, 12 beta-Epoxypukalide as a feeding deterrent in Phyllogorgia dilata.J. Nat. Prod. 25:2255-2265.

Erel, O. 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin. Biochem. 37: 277-285.

Fabricius, K., Alderslade, P. 2001. Soft Corals and Sea Fans: A Comprehensive Guide to the Tropical Shallow Water Genera of the Central West Pacific, the Indian Ocean and the Red Sea. Australian Institute of Marine Science, Townsville, Australia.

Falkowski, P.G., Raven, J.A. 1997. Aquatic Photosynthesis. Malden, MA: Blackwell Sci. 375 pp.

Fankboner, P. V., and R. G. B. Reid. 1981. Mass expulsion of zooxanthellae by heat stressed reef corals: a source of food for giant clams, Tridacna gigas. Experentia 37:251- 252.

Faulkner, D. J. 1998. Marine natural products. Nat. Prod. Rep. 2:113-159.

Faulkner, D.J. 2000. Highlights of marine natural products chemistry (1972-1999). Nat. Prod. Rep.17:1-6.

Faulkner, D.J. 2000. Marine pharmacology. Antonie Van Leeuwenhoek International J. Gen. Mol. Microbio. 77, 135-145.

Faulkner,D. J., Harper,M. K., Salomon, C. E., and Schmidt, E.W. 1999. Localisation of bioactive metabolites in marine sponges. Mem. Queensl. Mus. 44:167–173.

Fenical, W. 1987. Marine soft corals of the genus Pseudopterogorgia: a resource for novel anti-inflammatory diterpenoids J. Nat. Prod. 50:1001-1008.

139 Fenical, W., and Pawlik, J. R. 1991. Defensive properties of secondary metabolites from the Caribbean gorgonian coral Erythropodium caribaeorum. Mar. Ecol. Prog. Ser.75:1-8.

Ferns, T. 2005. Elucidation of pseudopterosin biosynthesis: oxidations of Eragorgiaene and the intermediacy of the seco-pseudopterosins. Florida Atlantic University, Ph.D. Dissertation.

Fitt, W.K., Warner, M.E. 1995. Bleaching patterns of four species of Caribbean reef corals. Bio. Bull. 187:298–307.

Fitt, W. K., Brown, B. E., Warner, M. E., Dunne, R. P. 2001. Coral bleaching: interpretation of thermal tolerance limits and thermal thresholds in tropical corals. Coral Reefs. 20:51 -65.

Fenical, W. 1996. Marine biodiversity and the medicine cabinet: the status of new drugs from marine organisms. Oceanography 9:23-27.

Floyd, R.A. 1999. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc. Soc. Exp. Biol. Med. 222:236–45.

Fordyce, J.A. 2006. The evolutionary consequences of ecological interactions mediated through phenotypic plasticity. J. Exp. Biol. 209: 2377-2383.

Franklin DJ, Hoegh-Guldberg O, Jones RJ, Berges JA (2004) Cell death and degeneration in the symbiotic dinoflagellates of the coral Stylophora pistillata during bleaching. Mar Ecol Prog Ser 272:117–130.

Freyer, A.J., Patil, A.D. Killmer, L., Zuber, G., Myers, C., Johnson, R.K. 1997. Rigidone, a sesquiterpene o-quinone from the gorgonian Pseudopterogorgia rigida. J. Nat. Prod. 60: 309-311.

Fridovich, I. 1975. Superoxide dismutases. Science 44:147-59.

Fridovich, I. 1998. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201:1203–9.

Fryer, M.J. 1993. Evidence for the photoprotective effects of vitamin E. Photochem. Photobiol. 58:304–312.

Fujimoto, K., Kaneda, T. 1984. Separation of antioxigenic (antioxidant) compounds from marine algae. Hydrobiologia 116/117: 111–113.

Gao, Q., Zhang, L.X. 2008. Ultraviolet-B-induced oxidative stress and antioxidant defense system responses in ascorbate-deficient vtc1 mutants of Arabidopsis thaliana. J. Plant Physiol. 8:138–148.

Gardner, T.A., Cote, I.M., Gill, J.A., Grant, A., Watkinson, A.R. 2003. Long-term

140 region-wide declines in Caribbean corals. Science 301:958-960.

Gates, R. 1990. Seawater temperature and sublethal coral bleaching in Jamaica. Coral Reefs 8:192-197.

Gates, R.D., Edmunds, P.J. 1999. The physiological mechanisms of acclimatization in tropical reef corals. Am. Zool. 39:30–43.

Gerhart, D.J., Rittschof, D., Mayo, S.W. 1988. Chemical ecology and the search for marine antifoulants. Studies of a predator-prey symbiosis. J. Chem. Ecol.14:1905-1917.

Giamoustaris, A., Mithen, R. 1995. The effect of modifying the glucosinolate content of leaves of oilseed rape on its interaction with specialist and generalist pests. Ann. Appl. Biol.126:347–363

Gil-Turnes,M. S. and Fenical,W. 1992. Embryos of Homarus americanus are protected by epibiotic bacteria. Biol. Bull. 182:105–108.

Gil-Turnes, M. S., Hay, M. E., Fenical, W. 1989. Symbiotic marine bacteria chemically defend crustacean embryos from a pathogenic fungus. Science. 246:116–118.

Gleason, D. F., Wellington, G.M. 1993. Ultraviolet radiation induces coral bleaching: Evidence from field experiments. Nature 365:836-838.

Glebova, N.O., Ginty, D.D. 2005. Growth and survival signals controlling sympathetic nervous system development. Annu. Rev. Neurosci. 28:191–222.

Gleeson, M. W., Strong, A. E. 1995. Applying MCSST to coral reef bleaching. Adv. Space Res. 16:151-154.

Glynn, P. 1996. Coral reef bleaching: facts, hypotheses, and implications. Global Change Biology 2:495-509.

Glynn, P. 1991. Coral reef bleaching in the 1980s and possible connections with global warming. Trends Ecol. Evol. 6:175-179.

Glynn, P. and L. D'Croz. 1990. Experimental evidence for high temperature stress as the cause of El- Niño-coincident coral mortality. Coral Reefs 8:181-191.

Glynn, P. 1988. El Niño warming, coral mortality, and reef framework destruction by echinoid bioerosion in the eastern Pacific. Galaxea 7:129-160.

Goenaga, C. and M. Canals. 1990. Island-wide coral bleaching in Puerto Rico. Carib. J. Sci. 26:171-175.

141 Goffredo, S., Lakser, H.R. 2008. An adaptive management approach to an octocoral fishery based on the Beverton-Holt model. Corals Reefs. DOI 10.1007/s00338-008-0391- 6.

Goldstein DS, Robertson D, Esler M, Straus SE, Eisenhofer G. 2002. Dysautonomias: clinical disorders of the autonomic nervous system. Ann. Intern. Med. 137: 753–63.

Goreau, T. F. and N. I. Goreau. 1960. Distribution of labelled carbon in reef building corals with and without zooxanthellae. Science 181:668-669.

Goreau, T. J., R. L. Hayes, and A. E. Strong. 1997. Tracking South Pacific coral reef bleaching by satellite and field observations. Proceedings of the 8th International Coral Reef Symposium 2:1491-1494.

Goreau, T. J. and R. L. Hayes. 1994. Coral bleaching and ocean "hot spots." Ambio 23:176-180.

Goreau, T. J., R. L. Hayes, J. W. Clark, D. J. Basta, and C. N. Robertson. 1993. Elevated sea surface temperatures correlate with Caribbean coral reef bleaching. Pages 225-255 in Geyer, R. A. (ed.). A Global Warming Forum: Scientific, Economic, and Legal Overview. CRC Press, Ann Arbor, Michigan.

Goreau, T. J. 1992. Bleaching and reef community change in Jamaica:1951-1991. American Zoologist 32:683-695.

Goreau, T. J. 1990. Coral bleaching in Jamaica. Nature 343:417.

Goreau, T. J. and A. H. Macfarlane. 1990. Reduced growth rate of Montastrea annularis following the 1987-1988 coral bleaching event. Coral Reefs 8:211-216.

Goulet, T.L., Coffroth, M.A. 2003. Stability of an octocoral-algal symbiosis over time and space. Mar. Ecol. Prog. Ser. 250: 117–124.

Goulet, T.L., Coffroth, M.A. 2004. The genetic identity of dinoflagellate symbionts in Caribbean octocorals. Coral Reefs 23:465–472.

Goulet, T.L., Cook, C.B., Goulet, D. 2005. Effect of short-term exposure to elevated temperatures and light levels on photosynthesis of different host-symbiont combinations in the Aiptasia pallida Symbiodinium symbiosis. Limnol Oceanogr 50:1490–1498.

Goulet, T.L.2006. Most corals may not change their symbionts. Mar. Ecol. Prog. Ser. 321:1–7.

Goulet, T.J., LaJeunesse, T. C, Fabricus, K.E. (2008) Symbiont specificity and bleaching susceptibility among soft corals in the 1998 Great Barrier Reef mass coral bleaching event. Marine Biology. 154: 795-804.

142 Grace, S., Logan, B.A. 2000. Energy dissipation and radical scavenging by plant phenylpropanoid pathway. Phil. Tran. Royal Soc. London B. 355: 1499-1510.

Greenlund, L.J., Deckwerth, T.L., Johnson, E.M. Jr. 1995. Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death, Neuron 14:303–315.

Grigg, R. W., J. J. Polovina, and M. J. Atkinson. 1984. Model of a coral reef ecosystem. III. Resource limitation, community regulation, fisheries yield and resource management. Coral Reefs 3:23-27.

Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Jiang,Y. 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutrition Research 23:1719–1726.

Gutierrez-Rodriguez, C., Lasker, H.R. 2004. Reproductive biology, development, and planula behavior in the Carribean gorgonian Pseudopterogorgia elisabethae. Invert. Biol. 123:53-66.

Gutierrez-Rodriguez, C., Lasker, H.R. 2004. Microsatellite variation reveals high levels of genetic variability and population structure in the gorgonian coral Pseudopterogorgia elisabethae across the Bahamas. Mol. Ecol. 13: 2211–2221.

Gutierrez-Rodriguez, C., Lasker, H.R. 2004. Isolation and characterization of microsatellite loci in the Caribbean gorgonian Pseudopterogorgia elisabethae. Mol. Ecol. Notes 4: 153–155.

Halliwell B, Gutteridge J. 1999. Free Radicals in Biology and Medicine, 3nd edn. Clarendon Press: Oxford.

Haraguchi, H., Saito, T., Okamura, N., Yagi, A. 1995. Inhibition of lipid peroxidation and superoxide generation by diterpenoids from Rosmarinus officinalis. Planta Med. 61: 333–336.

Harvell, C.D., Kim, K., Burkholder, J.M., Colwell, R.R., Epstein, P.R., Grimes, D.J., Hofmann, E.E., Lipp, E.K., Osterhaus, A.D ., Overstreet, R.M., Porter, J.W., Smith, G.W., Vasta, G.R. 1999. Emerging marine diseases-Climate links and anthropogenic factors. Science. 285:1505-1510.

Harvell, C.D., Mitchell, C.E., Ward, J.S., Altizer, S., Dobson, A.P., Ostfeld, R.S., Samuel, M.D. 2002. Climate warming and disease risks for terrestrial and marine biota. Science. 296: 2157 – 2162.

Harvell, C. D. and Suchanek, T.H. 1987. Partial predation on tropical gorgonians by Cyphoma gibbosum (Gastropoda).Mar. Ecol. Prog. Ser. 49:287-294.

143 Harvell, C. D., Fenical, W., Greene, C. H. 1988. Chemical and structural defenses of Caribbean gorgonians (Pseudopterogorgia spp.): Development of a field feeding assay. Mar. Ecol. Prog. Ser. 53:354-566.

Harvell, C. D., Fenical, W., Roussis, V., Ruesink, J.L., Griggs, C.C., Greene, C.H. 1993. Local and geographic variation in the defensive chemistry of a West Indian gorgonian coral (Briarium asbestinum). Mar. Ecol. Prog. Ser. 93:165-173.

Harvell, C. D., West, J. M., and Griggs, C. 1996. Chemical defense of embryos and larvae of a West Indian gorgonian coral, Briareum asbestinus. Inven. Reprod. Dev. 30:239–246.

Harvell, D., Kim, K., Quirolo, C., Weir, J., Smith, G. 2001. Coral bleaching and disease: contributors to 1998 mass mortality in Briareum asbestinum(Octocorallia, Gorgonacea). Hydrobiologia. 460:97 – 104.

Haugland, R.P. The Handbook—A Guide to Fluorescent Probes and Labeling Technologies (10th ed.). 2005. Invitrogen Corp.

Havaux, M., Eymery, F., Porfirova, S., Rey, P., Dörmann, P. 2005. Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana. Plant Cell 17:3451–3469.

Hay, M.E. and W. Fenical. 1996. Chemical ecology and marine biodiversity: insights and products. Oceanography 9:10-20.

Hayes, R. L., Goreau, N. I. 1998. The significance of emerging diseases in the tropical coral reef ecosystem. Rev. Biol. Trop. 5:173-185.

Hayes, R. L., Goreau, T.J. 1991. Tropical coral reef ecosystems as a harbinger of global warming. World Resource Review 3:306-322.

Hayes, R. L., Bush, T. 1990. Microscopic observations of recovery in the reef building scleractinian coral Montastrea annularis, after bleaching on a Cayman reef. Coral Reefs 5:201-204.

Hayes, R. L., W. C. Jaap, R. I. Wicklund, E. H. Williams, Goreau, T. J. 1990. collected testimonies present to the U. S. Senate Committee on Commerce, Science and Transportation.

Haygood, M.G., Davidson, S.K. 1997. Small-subunit rRNA genes and in situ hybridization with oligonucleotides specific for the bacterial symbionts in the larvae of the bryozoan Bugula neritina and proposal of Candidatus Endobugula sertula. Appl Environ Microbiol. 63:4612–461.

144 Haygood, M.G., Davidson, S.K. 1998. Bacterial symbionts of the bryostatin-producing bryozoan Bugula neritina. In: LeGal Y, Halvorson HO (eds) New developments in marine biotechnology. Plenum Press, New York. 281–284.

Haygood, M.G., Schmidt, E.W., Davidson, S. K., Faulkner, D. J. 1999. Microbial symbionts of marine invertebrates, opportunities for microbial biotechnology. J. Mol. Microbiol. Biotechnol. 1:33–43.

Hoegh-Guldberg, O., Smith, G. J. 1989. Light, salinity, and temperature and the population density, metabolisms and export of zooxanthellae from Stylophora pistillata and Seriatopora hystrix. J. Exper. Mar. Biol. Ecol. 129:279-303.

Hoegh-Guldberg, O., Salvat, B.S. 1995. Periodic mass-bleaching and elevated sea temperatures: Bleaching of outer reef slope communities in Moorea, French Polynesia. Mar. Ecol. Prog. Ser.121: 181–190.

Hoegh-Guldberg, O. 1999. Climate change, coral bleaching and the future of the world's coral reefs. Marine & Freshwater Research 50:839-866.

Hoegh-Guldberg O. 2005. Low coral cover in a high-CO2 world. J. Geophys. Res.

Horwitz, S. B.,Fant, J., Schiff, P. B. 1979. Promotion of microtubule assembly in vitro by taxol. Nature. 277: 665.

Hughes, T.P. 1994. Catastrophes, phase shifts, and large-scale degradation of a Caribbean coral reef. Science 265:1547–1551.

Hutchings, P. A. 1986. Biological destruction of coral reefs: a review. Coral Reefs 4:239- 252.

IPCC. 1998. The Regional Impacts of Climate Change: An Assessment of Vulnerability. Intergovernmental Panel on Climate Change. Watson, R. T., M.C. Zinyowera, and R. H. Moss (eds.). Cambridge University Press, New York.

IPCC. 1995. Climate Change 1995: The Science of Climate Change. Intergovernmental Panel on Climate Change. Houghton, J.T., L.G. Meira Filho, B.A. Callender, N. Harris, A. Kattenberg, and K. Maskell (eds.). Cambridge University Press, UK.

ISRS. 1998. Statement on global coral bleaching in 1997-1998. The International Society for Reef Studies (ISRS), consisting of more than 750 members in more than 50 countries, was founded in 1981 for the purpose of promoting the production and dissemination of scientific knowledge and understanding of coral reefs, both living and fossil.

ITMEMS. 1998. ITMEMS statement on coral bleaching is a product of The International Coral Reef Initiative (ICRI).

145 Iglesias-Prieto, R., Trench, R. K. 1997. Acclimation and adaptation to irradiance in symbiotic dinoflagellates. II. Response of chlorophyll-protein complexes to different photon-flux densities. Mar. Biol. 130:23 -33.

Iglesias-Prieto, R., Matta, W. A., Robins, W. A.,Trench, R. K. 1992. Photosynthetic response to elevated temperature in the symbiotic dinoflagellate Symbiodinium microadriaticum in culture. Proc. Natl. Acad. Sci. 89:10302 10305.

Iglesias-Prieto, R., Govind, N. S., Trench, R. K. 1993. Isolation and characterization of three membrane-bound chlorophyll-protein complexes from four dinoflagellate species. Philos. Trans. R. Soc. Lond. B Biol. Sci. 340:381 -392.

Jaakola, L., Määttä-Riihinen, K. 2004. Activation of flavonoid biosynthesis by solar radiation in bilberry (Vaccinium myrtillus L.) leaves. Planta 218:721–728.

Jaap, W. C. 1985. An epidemic zooxanthellae expulsion during 1983 in the lower Florida Keys coral reefs: hyperthermic etiology. Proceedings of the 5th International Coral Reef Symposium 6:143-148.

Jackson, J.B.C. 1997. Reefs since Columbus. Coral Reefs. 16:S23-S32.

Jackson, J.B.C. 2001. What was natural in the coastal oceans? PNAS 98:5411-5418.

Jameson, S. C., J. W. McManus, and M. D. Spalding. 1995. State of the Reefs: Regional and Global Perspectives. U. S. Department of State, Washington, D.C.

Jensen, P.R., Harvell, C.D., Wirtz, K., Fenical, W. 1996. The incidence of antimicrobial activity among Caribbean gorgonians. Mar. Biol. 125: 411-419.

Jokiel, P. L., York, R.H. 1982. Solar ultraviolet photobiology of the reef coral Pocillopora damicornis and symbiont zooxanthellae. Bull. Mar. Sci. 32:301-315.

Jokiel, P. and S. L. Coles. 1990. Response of Hawaiian and other Indo-Pacific corals to elevated temperatures. Coral Reefs 8:155-162.

Jones, R.J., Kildea, T., Hoegh-Guldberg, O. 1999. PAM chlorophyll fluorometry: a new in situ technique for stress assessment in scleractinian corals, used to examine the effects of cyanide from cyanide fishing, Mar. Pollut. Bull. 38: 864–874.

Jones, R., Hoegh-Guldberg, O., Larkum, A. W. D., Schreiber, U. 1998. Temperature induced bleaching of corals begins with impairment of dark metabolism in zooxanthellae. Plant Cell Environ. 21:1219 -1230.

Jones, R. J., Ward, S., Yang Amri, A., Hoegh-Guldberg, O. 2000. Changes in quantum efficiency of Photosystem II of symbiotic dinoflagellates of corals after heat stress, and of

146 bleached corals sampled after the 1998 Great Barrier Reef ass Bleaching event. Mar. Freshw. Res. 51:63 -71.

Karban, R., Agrawal, A.A. 2002. Herbivore offense. Annu. Rev. Ecol. Syst. 33:641–664.

Kellner, R.L.L., Dettner, K. 1995. Allocation of pederin during lifetime of Paederus rove beetles (Coleoptera: Staphylinidae): evidence for polymorphism of hemolymph toxin. J. Chem. Ecol. 21:1719–1733.

Kellner, R.L.L. 2001. Suppression of pederin biosynthesis through antibiotic elimination of endosymbionts in Paederus sabaeus. J Insect Physiol. 47:475–483.

Kellner, R.L.L. 2002. Molecular identification of an endosymbiotic bacterium associated with pederin biosynthesis in Paederus sabaeus (Coleoptera: Staphylinidae). Insect Biochem. Mol. Biol. 32:389–395.

Kerr, J.F., A.H. Wyllie, and A.R. Currie. 1972. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26:239– 257.

Kerr, R. A. 1999. Big El Niños ride the back of slower climate change. Science 283: 1108-1109.

Kilsch, M., Hader, D.P. 2008. Mycosporine-Like Amino Acids and Marine Toxins - The Common and the Different. Mar. Drugs. 6:147-163.

Kim, K.,Kim, P.D., Alker, A.P., Harvell, C.D. 2000. Chemical resisitance of gorgonian corals against fungal infection. Mar Biol. 137:393-401.

Kim, K. 1994. Antimicrobial activity in gorgonian corals (Coelenterata, Octocorallia). Coral Reefs. 13:75-80.

Kinzie, R. 1973. The zonation of West Indian gorgonians. Bull. Mar. Sci. 23: 93-155.

Kinzie, R.A. 1974. Experimental infection of aposymbiotic gorgonian polyps with zooxanthellae. J. Exp. Mar. Biol. Ecol. 15:335–342.

Kinzie, R. A. 1999. Sex, symbiosis and coral reef communities.Amer. Zool. 39:80-91.

Kirk, N.L., Ward, J.R., Coffroth, M.A. 2005. Stable Symbiodinium composition in the sea fan Gorgonia ventalina during temperature and disease stress. Biol. Bull. 209:227–234.

Kirkland, R.A., Franklin, J.L. 2003. Prooxidant Effects of NGF Withdrawal and MEK Inhibition in Sympathetic Neurons. Antiox. Redox Sig. 5:635-639.

147 Kirkland, R.A., Saavedra, G.M., Franklin, J.L. 2007. Rapid Activation of Antioxidant Defenses by Nerve Growth Factor Suppresses Reactive Oxygen Species during Neuronal Apoptosis: Evidence for a Role in Cytochrome c Redistribution. J. Neurosci. 27: 11315- 11326.

Kitajima, M., Butler, W.L. 1975. Quenching of chlorophyll fluorescence and primary photochemistry in chloroplasts by dibromothymoquinone. Biochim. Biophys. Acta 376: 105–115.

Kleppel, G.S., Dodge, R.E., Reese, C.J. 1989. Changes in pigmentation associated with the bleaching of stony corals. Limnology and Oceanography 34:1331-1335.

Kobayashi, J., Ishibashi, M. 1993. Bioactive metabolites of symbiotic marine microorganisms. Chem. Rev. 93:1753–1769.

Koh, L.L., Goh, N.K.C., Chou, L.M., Tan, Y.W. 2000. Chemical and physical defense of Singapore gorgonians (Octocorallia: Gorgonacae). J. Exp. Mar. Biol. Ecol. 251:103-115.

Kokke, W.C.M.C., Epstein, S., Look, S.A., Rau, G.H., Fenical, W., Djerrasi, C. 1980. On the origin of terpenes in symbiotic associations between marine invertebrates and algae (zooxanthellae). J. Biol. Chem. 259:8168-8173.

Körner, C. 1999. Alpine Plant Life. Functional Plant Ecology of High Mountain Ecosystems., Springer, Berlin.

Kuda, T., Tsunekawa, M., Goto, H., Araki, Y. 2005. Antioxidant properties of four edible algae harvested in the Noto Peninsula, Japan, J. Food Comp. Anal. 18:625–633.

La Barre, S.C., Coll, J.C., Sammarco, P.W. 1986. Competitive strategies of soft corals (Coelenterata: Octocorallia). III. Spacing and aggressive interactions between alcyonaceans. Mar. Ecol. Prog. Ser. 28:147–156.

LaJeunesse, T. C. 2002. Diversity and community structure of symbiotic dinoflagellates from Caribbean coral reefs. Mar. Biol. 141: 387–400.

LaJeunesse, T. C., Loh, W. K. W., Van Woesik, R., Hoegh-Guldberg, O., Schmidt, G. W., Fitt, W. K. 2003. Low symbiont diversity in southern Great Barrier Reef corals, relative to those of the Caribbean. Limnol. Oceanogr. 48: 2046–54.

LaJeunesse, T. C., Thornhill, D. J., Cox, E. F., Stanton, F. G., Fitt, W. K., Schmidt, G. W. 2004. High diversity and host specificity observed among symbiotic dinoflagellates in reef coral communities from Hawaii. Coral Reefs 23: 596–603.

Lapointe, B. E. (1999). Simultaneous top-down and bottom-up forces control macroalgal blooms on coral reefs (reply to the comment by Hughes et al.). Limnol. Oceanogr. 44, 1586–1592.

148

Lasker, H.R., Coffroth, M.A. 1983. Octocoral distributions at Carrie Bow Cay, Belize. Mar. Ecol. Prog. Ser. 13:21-28.

Lasker, H.R. 1985. Prey preferences and prowsing pressures of the butterfly fish Chaetodon capistratus on Caribbean gorgonians. Mar. Ecol. Progr. Ser. 21: 213-220.

Lasker HR, Peters EC, Coffroth MA (1984) Bleaching of reef coelenterates in the San- Blas Islands, Panama. Coral Reefs, 3, 183–190.

Lasker HR. 2003. Zooxanthella densities within a Caribbean octocoral during bleaching and nonbleaching years. Coral Reefs 22(1):23-26.

Leder, J. J., A. M. Szmant, and P.K. Swart. 1991. The effect of prolonged "bleaching" on skeletal banding and stable isotope composition in Montastrea annularis. Preliminary observations. Coral Reefs 10:19-27.

León, J., Rojo, E., Sánchez-Serrano, J.J. 2001. Wound signalling in plants. J. Exp. Bot. 52:1–9.

Lesser, M. P., Stochaj, W. R.,Tapley, D. W., Shick, J. M. 1990. Bleaching in coral reef anthozoans: Effects of irradiance, ultraviolet radiation and temperature on the activities of protective enzymes against active oxygen. Coral Reefs 8:225-232.

Lesser, M.P. 1996. Elevated temperatures and ultraviolet radiation cause oxidative stress and inhibit photosynthesis in symbiotic dinoflagellates, Limnol. Oceanogr. 41:271–283.

Lesser, M.P. 1997. Oxydative stress causes coral bleaching during exposure to elevated temperature, Coral Reefs. 16:187–192.

Lesser, M.P. 2006. Oxidative stress in marine environments: Biochemistry and Physiological Ecology. Annu. Rev. Physiol. 68:253–78.

Leuzinger, S., Anthony, K.R.N., Willis, B.L. 2003. Reproductive energy investment in corals: scaling with module size. Oecologia136: 524–531.

Lim, S.N., Cheung, P.C.K., Ooi, V.E.C., Ang, P.O., 2002. Evaluation of antioxidative activity of extracts from a brown seaweed, Sargassum siliquastrum. J. Ag. Food Chem. 50: 3862-3866.

Lindquist, N., Hay, M. E. 1995. Can small rare prey be chemically defended? The case for marine larvae. Ecology. 76:1347–1358.

Lindquist, N. 2002. Chemical Defense of Early Life Stages of Benthic Marine Invertebrates. J. Chem Ecol. 28:1987-2000.

149

Look, S.A.,Burch, M.T., Fenical, W. Qi-tai, Z., Clardy, J. 1985. Kallolide A, antiinflammatory diterpenoid and related lactones from Caribbean octocoral Pseudopterogorgia kallos. J. Org. Chem. 50:5741-5746.

Look, S.A., Fenical, W., Matsumoto G., Clardy, J. 1986. The pseudopterosins: a new class of anti-inflammatory and analgesic diterpene pentosides from the marine sea whip Pseudopterogorgia elisabethae. J. Org. Chem. 51:5140-5145.

Lopanik, N., Lindquist, N., Targett, N. 2004. Potent cytotoxins produced by a microbial symbiont protect host larvae from predation. Oecologia 139:131–139.

Lorenz, E. N. 1963. Deterministic nonperiodic flow. Journal of Atmospheric Science 20:130-141.

Low PA. 2002. Autonomic neuropathies. Curr. Opin. Neurol. 15: 605–9.

Loya, Y.,Akai, K.S., Amazato, K.Y., Akano, Y.N., Ambali, H.S., Van Woesik, R. V. 2001. Coral bleaching: The winners and the losers. Ecol. Lett. 4: 122–131.

Lupınkova, L., Komenda, J. 2004. Oxidative modification of the photosystem II D1 protein by reactive oxtgen species: From isolated protein to cyanobacterial cells. Photochem. Photobiol. 79:152–62.

Luthra R., Luthra, P.M., Kumar, S. 1995. Redefined role of mevalonate-isoprenoid pathway in terpenoid biosynthesis in higher plants. Current Sci. 76:133-135.

Maia, L.F., Epifano, R.D., Eve, T., Fenical, W. 1999. New fish feeding deterrents, including novel sesquiterpenoid Heterogorgiolide, from the Brazilian gorgonian Heterogorgia uatumani. J. Nat. Prod. 62:1322-1324.

Mann, M.E., R. S. Bradley, and M. K. Hughes. 1998. Global-scale temperature patterns and climate forcing over the past six centuries. Nature 392:779-787.

Mantua, N. J. , S. R. Hare, Y. Zhang, J. M. Wallace, and R. C. Francis. 1997. A Pacific interdecadal climate oscillation with impacts on salmon production. Bull. Amer. Met. Soc. 78:1069-1079.

Matsukawa, R., Dubinsky, Z., Kishimoto, E., Masak, K., Masuda, Y., Takeuchi, T., Chihara, M., Yamamoto, Y., Niki, E., Karube, I., 1997. A comparison of screening methods for antioxidant activity in seaweeds. J. Appl. Phycol. 9:29-35.

Mayer, A.M.S., Jacobson, P.B., Fenical, W., Jacobs, R.S., Glaser, K.B. 1998. Pharmacological characterization of the the pseudopterosins: novel anti-inflammatory natural products isolated from the Caribbean soft coral Pseudopterogorgia elisabethae. Life Sci. 62:401–407.

150

McAllister, D. E. 1988. Environmental, economic and social costs of coral reef destruction in the Philippines. Galaxea 7:161-178.

McClintock, J. B., Baker, B. J. (eds.). Marine Chemical Ecology. 2001, CRC Press, Boca Raton, Florida.

McEnroe, F.J., Fenical, W. 1978. Structures and synthesis of some new antibacterial sesquiterpenoids from the gorgonian coral Pseudopterogorgia rigida. 1978. Tetrahedron 34:1661-4.

McGarvey, D.J., Croteau, R. 1995. Terpenoid metabolism. Plant Cell 7: 1015-1026.

McPhaden, M. J. 1999. Genesis and evolution of the 1997-98 El Niños. Science 283:950- 954.

Michalek-Wagner, K., Bourne, D.J. and Bowden, B.F. 2001. The effects of different strains of zooxanthellae on the secondary-metabolite chemistry and development of the soft-coral host Lobophytum compactum. Mar. Bio. 138:753-760.

Miquel, J. 2001. Nutrition and ageing. Public Health Nutr. 4:1385–8.

Montgomery, R. S. and A. E. Strong. 1994. Coral bleaching threatens oceans, life. EOS 75:145-147.

Mukai, K., Kohno, Y., Ishizu, K. 1988. Kinetic study of the reaction between vitamin E radical and alkyl hydroperoxides in solution. Biochem. Biophys. Res. Commun. 155:1046–1050.

Mukherjee, M. S. 2005. Diterpene Biosynthesis in Early Ontogenetic Stages of Pseudopterogorgia elisabethae and Pseudopterogorgia bipinnata. Florida Atlantic University, MS Thesis.

Munné-Bosch, S., Alegre, L. 2001. Subcellular compartmentation of the diterpene carnosic acid and its derivatives in the leaves of rosemary. Plant Physiol. 125:1094-1102.

Munné-Bosch, S., Alegre, L. 2002. Interplay between ascorbic acid and lipophilic antioxidant defences in chloroplasts of water-stressed Arabidopsis plants. FEBS Lett. 524:145–148.

Munro, J. L. 1996. The scope of tropical reef fisheries and their management. Pages 1-14 in Polunin, N. V. C. and C. M. Roberts. Reef Fisheries. Chapman and Hall, London.

Muscatine L., Pool R.R. & Cernichiari E. 1972 Some factors influencing selective release of soluble organic material by zooxanthellae from reef corals. Marine Biology 13, 298–308.

151 Muscatine, L. 1973. Nutrition of corals. Pages 77-115 in Biology and Geology of Coral Reefs, Vol. 2, Jones, O. A., and R. Endean (eds.). Academic Press, New York.

Muscatine, L., Porter, J.W. 1977. Reef corals - mutualistic symbioses adapted to nutrient- poor environments. Bioscience 27:454–460

Muscatine, L. 1990. The Role of Sym-biotic Algae in Carbon and Energy Flux in Reef Corals. In: Dubinsky Z., editor. Coral Reefs Ecosystems of the World. Vol. 25. Elsevier: pp. 75–87.

Mydlarz, L.D., Jacobs, R.S., Boehnlein, J., Kerr, R.G. 2003. Pseudopterosin Biosynthesis in Symbiodinium sp., the Dinoflagellate Symbiont of Pseudopterogorgia elisabethae. Chem. & Bio. 10:1-20.

Nakagawa, H., Murata, M., Tachibana, K., Shiba, T. 1998. Screening of epiphytic dinoflagellates for radical scavenging and cytotoxic activities. Phycol Res 46:9-12.

Navarro, A., Gomez, C., Sanchez-Pino, M.J., Gonzalez, H., Bandez, M.J., Boveris, A.D., Boveris, A. 2005. Vitamin E at high doses improves survival, neurological performance, and brain mitochondrial function in aging male mice. Am. J. Physiol. Regul. Integ. Comp. Physiol. 289:R1392–9.

Newman, D.J., Cragg, G.M. 2004. Marine natural products and related compounds in clinical and advanced preclinical trials. J. Nat. Prod. 67:1216-1238.

Newman, R.A. 1992. Adaptive plasticity in amphibian metamorphosis: what type of phenotypic variation is adaptive, and what are the costs of such plasticity. BioScience 42: 671–678.

Nishiyama, Y., Yamamoto, H., Allakhverdiev, S.I., Inaba, M., Yokota, A. 2001. Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery. EMBO. J. 20:5587–94.

Noctor, G., Foyer, C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 49:249–279.

Ogden, J. C. 1977. Carbonate-sediment production by parrot fish and sea urchins on Caribbean reefs. Pages 281-288 in S. H. Frost, M. P. Weiss and J. B. S. (eds.). Reefs and Related Carbonates - Ecology and Sedimentology. The American Association of Petroleum Geologists, Tulsa, Oklahoma.

O’Neal, W., Pawlik, J.R. 2002. A reappraisal of the chemical and physical defenses of Caribbean gorgonian corals against predatory fishes. Mar. Ecol. Prog. Ser. 240:117-126.

Opresko, D. 1973. Abundance and distribution of shallow water gorgonians in the area of Miami, Florida. Bull. Mar. Sci. 23: 533-558.

152 Orr, J.C., Fabry, V.J., Aumont, O., Bopp, L., Doney, S.C. 2005. Anthropogenic ocean acidification over the twenty-first century and its impact on calcifying organisms. Nature 437:681–86.

Ostrander, G.K., Armstrong, K.M., Knobbe, E.T., Gerace, D., Scully, E.P. 2000. Rapid transition in the structure of a coral reef community: The effects of coral bleaching and physical disturbance. Proc. Nat. Acad. Sci. 97:5297-5302.

Papastephanou, C., Anderson, D.G. 1982. Crassin acetate biosynthesis in a cell-free homogenate of zooxanthellae from Pseudoplexaura porosa (Houttyun): Implications to the symbiotic process. Comp. Biochem. Physiol. 73:617-624.

Pansarasa, O., Bertorelli, L., Vecchiet, J., Felzani, G., Marzatico, F. 1999. Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle. Free Radic. Biol. Med. 27:617–622.

Paul, V.J. 1992. Chemical defenses of benthic marine invertebrates. In: Paul VJ (ed) Ecological roles of marine natural products. Comstock Publishing Associates, Ithaca, New York. 164-188.

Paul V.J., Van Alstyne, K.L. 1992. Activation of chemical defense in the tropical green algae Halimeda spp. J. Exp. Mar. Biol. Ecol. 160:191–203.

Paul, V.J., Puglisi, M.P. 2004. Chemical mediation of interactions among marine organisms. Nat. Prod. Rep. 21:189 209.

Pawlik, J.R., Burch, M.T., Fenical, W. 1987. Patterns of chemical defenses among Caribbean gorgonian corals: a preliminary survey. J. Exp. Mar. Biol. Ecol. 108:55-66.

Pawlik, J.R. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar. Biol. Annu. Rev. 58:273-335.

Pawlik, J.R.1993.Marine invertebrate chemical defense. Chem. Rev. 93: 1911–1922.

Peters, R. L. 1988. Effects of global warming on species and habitats: an overview. Endangered Species UPDATE 5:1-8.

Pomponi, S.A. 1999. The bioprocess-technological potential of the sea. J. Biotechnol. 70:5–13.

Porter, J.W., Fitt, W.K., Spero, H. J., Rogers, C.S., White, M.W. 1989. Bleaching in reef corals: Physiological and stable isotopic responses. PNAS, 86: 9342-9346.

Porter, J.W., Meier, O.W. 1992. Quantification of loss and change in Floridian reef coral populations. American Zoologist 32:625-640.

153 Porter, J.W., Tougas, J.I. 2001. Reef ecosystems: threats to their biodiversity. In: S. Levin, Editor, Encyclopedia of Biodiversity, vol. 5, Academic Press. pp. 73–95.

Potts, D.C.1984. Natural selection in experimental populations of reef-building corals (). Evolution 38:1059–1078.

Prior, R.L., Wu, X., Schaich, K. J. 2005. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Ag. Food Chem. 53:4290–4302.

Proksch, P., Edrada, R.A., Edel, R. 2002. Drugs from the seas – current status and microbiological implications. App. Microbio. Biotech. 59:125-134.

Putcha, G.V., Deshmukh, M., Johnson, E.M.Jr. 1999. Bax translocation is a critical event in neuronal apoptosis: regulation by neuroprotectants, Bcl-2, and caspases. J Neurosci. 19:7476–7485.

Reaka-Kudla, M. L. 1996. The global biodiversity of coral reefs: a comparison with rainforests. Pages 83-108 in M. L. Reaka-Kudla, D. E. Wilson, and E. O. Wilson, (eds.), Biodiversity II: Understanding and Protecting Our Natural Resources. Joseph Henry/National Academy Press, Washington, D.C.

Reaka-Kudla, M. L., J.S. Feingold, and P. W. Glynn. 1996. Experimental studies of rapid bioerosion of coral reefs in the Galapagos Islands. Coral Reefs 15:101-107.

Rice-Evans, C.A., Miller, N.J., Paganga, G. 1997. Antioxidant properties of phenolic compounds. Trends Plant Sci. 2: 152-159.

Richheimer, S.L., Bailey, D.T., Bernart, M.W., Kent, M., Vininski, J.V., Anderson, L.D. 1999. Antioxidant activity and oxidative degradation of phenolic compounds isolated from rosemary. Recent Res. Dev. Oil Chem. 3: 45-58.

Richier, S., Furla, P., Plantivaux, A., Merle, P.L., Allemand, D. 2005. Symbiosis-induced adaptation to oxidative stress. J. Exp. Biology 208:277–285.

Richmond, R. H. 1993. Coral reefs: present problems and future concerns resulting from anthropogenic disturbance. American Zoologist 33:524-536.

Robinson, K.M., Janes, M.S., Pehar, M., Monette, J.S., Ross, M.F., Hagen, T.M., Murphy, M.P., Beckman, J.S. 2006. Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc. Natl. Acad. Sci. 103:15038–15043.

Rodríguez, A. D. 1995. The natural products chemistry of West Indian gorgonian octocorals. Tetrahedron. 51:4571-4618.

154 Rodríguez, A. D., Soto, J. J. 1996. A Novel Norditerpene from the Caribbean Sea Plume Pseudopterogorgia acerosa (Pallas). J. Org. Chem. 61:4487-4490.

Rodríguez, A. D., Shi, J.-G. 1998. The First Cembrane-Pseudopterane Cycloisomerization. J. Org. Chem. 63:420-421.

Rodríguez, A. D., Soto, J. J. 1998. Pseudopterane and Norcembrane Diterpenoids from the Caribbean Sea Plume Pseudopterogorgia acerosa. J. Nat Prod. 61:401-404.

Rodriguez, A.D., Shi, J.G., Huang, S. 1999. Highly Oxygenated Pseudopterane and Cembranolide Diterpenes from the Caribbean Sea Feather Pseudopterogorgia bipinnata. J. Nat. Prod. 62:1228-1237.

Rodriguez, A.D., Shi, Y.-P. 2000. New Metabolites from the West Indian Sea Feather Pseudopterogorgia bipinnata. J. Nat. Prod. 63:1548- 1550.

Rodriguez, A.D., Shi, Y.-P. 2000. Verrillin: A Highly Oxygenated Marine Diterpene Based on the Novel Verrillane Carbon Skeleton. J. Org. Chem. 65:5839-5842.

Rodriguez, A.D., Shi, J.G., Shi, Y.P. 2000. Isolation, Structural Characterization, and Synthesis of a Naturally Occurring Bisfuranopseudopterane Ether: Biskallolide A. Evidence for a Carbocation Intermediate during the Facile Conversion of Kallolide A and Isokallolide A into Various Solvolysis Products. J. Org. Chem. 65:3192- 3199.

Rodriguez-Lanetty, M., Chang,S.J., Song, J.I. 2003. Specificity of two temperate dinoflagellate-anthozoan associations from the northwestern Pacific Ocean. Mar. Biol. 143:1193–1199.

Rodriguez-Lanetty, M., Krupp, D.A., Weis, V.M. 2004. Distinct ITS types of Symbiodinium in Clade C correlate with cnidarian/dinoflagellate specificity during onset of symbiosis. Mar. Ecol. Prog. Ser. 275:97–102.

Romano, S. L., Palumbi, S. R. 1996. Evolution of Scleractinian Corals Inferred from Molecular Systematics. Science 271:640-645.

Romano, S.L., Cairns, S.D. 2000. Molecular Phylogenetic Hypotheses for The Evolution of Scleractinian Corals. Bull. Mar. Sci. 67: 1043-1068.

Romero, F.J., Bosch-Morell, F., Romero, M.J., Jareno, E.J., Romero, B., Marin, N., Roma, J. 1998. Lipid peroxidation products and antioxidants in human disease. Environ. Health Perspect. 106:1229–1234.

Rouhi, M. 1995. Supply issues complicate trek of chemicals from sea to market. Sci. Eng. News. 20:42-44.

155 Ruzicka, L. 1953. The isoprene rule and the biogenesis of terpenic compounds. Experientia. 9:357-367.

Rowan, R., Knowlton, N. 1995. Intraspecific diversity and ecological zonation in coral– algal symbiosis. Proc Natl Acad Sci USA 92:2850–2853

Rowan R, Knowlton N, Baker A, Jara J (1997) Landscape ecology of algal symbionts creates variation in episodes of coral bleaching. Nature 388:265–269

Rowan, R. 2004. Coral bleaching - thermal adaptation in reef coral symbionts. Nature 430:742–742. Ruberto, G., Baratta, M.T., Biondi, D.M. and Amico, V., 2001. Antioxidant activity of extracts of the marine algal genus Cystoseira in a micellar model system. J. App.Phycol. 3:403-407..

Ruperez, P., Ahrazem, O. and Leal, J.A., 2002. Potential antioxidant capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J. Ag. Food Chem. 50: 840–845.

Ruzicka, L. 1953. The isoprene rule and the biogenesis of terpenic compounds. Experientia 9:357-367.

Sammarco P.W., Coll, J.C., La Barre, S., Willis, B. 1983. Competitve strategies of soft corals (Coelenterata: Octocorallia): Allelopathic effects on selected Scleractinian Corals. Coral Reefs. 1:173-178.

Sánchez, J.A., Zea, S., Diaz, J.M. 1997. Gorgonian communities of two contrasting environments from oceanic Caribbean . Bull. Mar. Sci. 61:61-72.

Sánchez, J.A., Diaz, J.M., Zea, S. 1998. Octocoral and distribution patterns on the barrier reef-complex of Providencia island, Southwestern Caribbean. Carib.J. Sci. 34:250-264.

Sánchez, J.A. 1999. Black coral-octocoral distribution patterns on a deep-water reef, Imelda bank, Caribbean sea, Colombia. Bull. Mar. Sci. 65: 215-225.

Sánchez, J. A., Mcfadden, C. S., France, S. C., Lasker, H. R. 2003. Molecular Phylogenetic analyses of shallow-water Caribbean octocorals. Marine Biology 142: 975- 987.

Sánchez, J.A, Aguilar, C., Dorado, D., Manrique, N. 2007. Phenotypic plasticity and morphological integration in a marine modular invertebrate. BMC Evolutionary Bio. 7:122.

Sano, M., Ernesto, M.S., Thomas, R.G., Klauber, M.R., Schafer, K., Grundman, M., Woodbury, P., Growdon, J., Cotman, C.W., Pfeiffer, E., Schneider, L.S. 1997. A

156 controlled trial of selegiline, α-tocopherol, or both as treatment for Alzheimer's disease. N. Engl. J. Med. 336:1216–1222.

Santos, S.R., Taylor, D.J., Coffroth, M.A. 2001.Genetic comparisons of freshly isolated versus cultured symbiotic dinoflagellates: implications for extrapolating to the intact symbiosis. J. Phycol. 37:900–912.

Santos, S.R., Gutierrez-Rodriguez, C., Coffroth, M,A. 2003. Symbiodinim sp. associations in the gorgonian Pseudopterogorgia elisabethae in the Bahamas: high levels of genetic variability and population structure in symbiotic dinoflagellates. Mar. Biol. 143:111–120.

Satoh, K., Kadofuku, T., Sakagami, H. 1997. Effect of Trolox, a synthetic analog of α- tocopherol, on cytotoxicity induced by UV irradiation and antioxidants. Anticancer Res. 17:2459–2464.

Schmitz, F.J., Lorance, E.D., Ciereszko, L.S.1969. Chemistry of coelenterates. Hydroxyancepsenolide A dilactone from octocoral Pterogorgia anceps. J. Org. Chem. 34:1989-1990.

Schubert, N., Garcia-Mendoza, E., Pacheco-Ruiz, I. 2006. Carotenoid composition of marine red algae. J. Phycol. 42: 1208-1216.

Selkoe, D.J. 2005. Defining Molecular Targets to Prevent Alzheimer Disease Arch Neurol. 62:192-195.

Shen, B., Jensen, R.G., Bohnert, H.J. 1997. Mannitol protects against oxidation by hydroxyl radicals. Plant Physiol. 115: 527-532.

Singh, N., Luthra, R., Sangwan, R.S., Thakur, R.S. 1989. Metabolism of monoterpenoids in aromatic plants. Curr. Res. Med. Plants. 11:174-197.

Slattery, M., McClintock, J.B., Heine, J.N. 1995. Chemical defenses in Antarctic soft corals: evidence for antifouling compounds. J. Exp. Mar. Biol. Ecol.190: 61-77.

Slattery, M., Hines, G. A., Starmer, J., Paul, V. J. 1999. Chemical signals in gametogenesis, spawning, and larval settlement and defense of the soft coral Sinularia polydactyla. Coral Reefs. 18: 75–84.

Spitaler, R., Schlorhaufer, P. D., Ellmerer, E. P., Merfort, I., Bortrnschlager, S., Stuppner, H., Zidorn, C. 2006. Altitudinal variation of secondary metabolite profiles in flowering heads of Arnica montana cv. ARBO. Phytochemistry 67:409–417.

157 Spitaler, R., Winkler, A., Lins, I., Yanar, S., Stuppner, H., Zidorn, C. 2008. Altitudinal Variation of Phenolic Contents in Flowering Heads of Arnica montana cv. ARBO: a 3- Year Comparison. J. Chem. Ecol. 34: 369-375.

Stadtman, E.R. 2004. Role of oxidant species in aging. Curr. Med. Chem. 11:1105–1112.

Standing, J.D., Hooper, I.R., Costlow, J.D. 1984. Inhibition and induction of barnacle settlement by natural products present in octocorals. J. Chem. Ecol.10: 823-834.

Stanley, G.D. 2003. The evolution of modern corals and their early history. Earth-Sci. Rev. 60:195–225.

Stierle, A.C., Cardellina, J.H., Singleton, F.L. 1988. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Experientia. 44:1021.

Strong, A. E., Goreau, T. J., Hayes, R.L. 1998. Ocean HotSpots and coral reef bleaching: January - July 1998. Reef Encounters 24:20-22.

Strong, A. E., Barrientos, C. S., Duda, C., Sapper, J. 1997. Improved satellite techniques for monitoring coral reef bleaching. Proc. 8th International Coral Reef Symposium 2:1495-1498.

Sutherland, K.P., Porter, J.W., Torres, C. 2004. Disease and immunity in Caribbean and Indo-Pacific zooxanthellate corals. Marine Ecology Progress Series. 266:273-302.

Szmant, A. M., Gassman, N.J. 1990. The effects of prolonged "bleaching" on the tissue biomass and reproduction of the reef coral, Montastrea annularis. Coral Reefs 8:217-224.

Takahama, U. and Oniki, T. 1997. A peroxidase/phenolics/ascorbate system can scavenge hydrogen peroxide in plant cells. Physiol. Plant. 101:845–852.

Targett, N. M., Bishop, S. S., McConnell, O. J., Yoder, J. A. 1983. Antifouling agents against the benthic marine diatom, Navicula salinicola. Homarine from the gorgonians Leptogorgia virgulata and L. setacea and analogs. J. Chem. Ecol. 9:817-829.

Thornhill, D.J., LaJeunesse, T.C., Kemp, D.W., Fitt, W.K., Schmidt, G.W. 2006. Multi- year, seasonal genotypic surveys of coral-algal symbioses reveal prevalent stability or post-bleaching reversion. Mar. Biol. 148:711-722.

Thornton, R.S., Kerr, R.G. 2002. Induction of pseudopterosin biosynthesis in the gorgonian Pseudopterogorgia elisabethae. J. Chem. Ecol. 28: 2083-2088.

Tinto, W. F., Chan, W. R., Reynolds, W. F., McLean, S. 1990. Tobagolide, a new pseudopterane diterpenoid of the octocoral Pseudopterogorgia acerosa. Tetrahedron Lett. 31:465-468.

158 Tinto, W. F., Lough, A. J., Reynolds, W. F., McLean, S. 1991. A pseudopterane metabolite of Pseudopterogorgia acerosa containing an unusually stable diepoxyfuran. Tetrahedron Lett. 32:4661-4664.

Tinto, W. F., John, L., Reynolds, W. F., McLean, S. 1991. Novel pseudopteranoids of Pseudopterogorgia acerosa. Tetrahedron. 47: 8679-8686.

Tinto, W. F., Laydoo, R. S., Miller, S. L., Reynolds, W. F., McLean, S. 1995. 11- Gorgiacerol, a new pseudopteranoid from Pseudopterogorgia acerosa. J. Nat. Prod. 58: 1975-1977.

Titlyanov, E.A., Titlyanova, T.V., Tsukahara, J., Van Woesik, R., Yamazoto, K. 1999. Experimental increase of zooxanthella density in the coral Stylophora pistillata elucidate adaptive mechanisms for zooxanthellae regulation. Symbiosis 26:347-362.

Todd, P. A., R. J. Ladle, N. J. I. Lewin-Koh, Chou, L.M. 2004. Genotype ×environment interactions in transplanted clones of the massive corals Favia speciosa and Diploastrea heliopora. Mar. Ecol. Prog. Ser. 271:167-182.

Trebst, A., Depka, B., Holländer-Czytko, H. 2002. A specific role for tocopherol and of chemical singlet oxygen quenchers in the maintenance of photosystem 2 structure and function in Chlamydomonas reinhardtii. FEBS Lett. 56:156–160.

Tutour, B.L., Benslimane, F., Gouleau, M.P., Gouygou, J.P., Saadan, B., Quemeneur, F., 1998. Antioxidant and pro-oxidant activities of the brown algae, Laminaria digitata, Himanthalia elongata, Fucus vesiculosus, Fucus serratus and Ascophyllum nosum. J. App.Phycol. 10: 121-129.

Ulstrup, K.E., Berkelmans, R., Ralph, P.J., van Oppen, M.J.H. 2006.Variation in bleaching sensitivity of two coral species across a latitudinal gradient on the Great Barrier Reef: the role of zooxanthellae. Mar Ecol Prog Ser 314:135–148

Vacelet, J., Donadey, C. 1977. Electron microscope study of the association between some sponges and bacteria. J. Exp. Mar. Biol. Ecol. 30: 301-314.

Van Alstyne. K.L., Wylie, C.R., Paul, V.J., Meyer, K. 1992. Antipredator defenses in tropical Pacific soft corals (Coelenterata, Octocorallia). 1. Sclerites as defenses against generalist carnivorous fishes. Bio. Bull. 182: 231-240.

Van Etten, C.H., Tookey, H.L. 1982. Chemistry and biological effects of glucosinolates. In: Rosenthal, G.A., Janzen, D.H. (eds) Herbivores: their interaction with secondary plant metabolites. Academic Press, New York, p 471–500.

Wang, H., Joseph, J.A. 1999. Quantifying cellular oxidative stress by dichlorofluorescein assay using microplate reader. Free Radic. Biol. Med. 27:612–616.

159 Watanabe, N., Yamamoto, K., Ihshikawa, H., Yagi, A., Sakata, K., Brinen, L. S., Clardy, J., 1993. New chlorophyll a related compounds isolated as antioxidants from marine bivalves.J. Nat. Prod. 53:305–317.

Warner, M., Fitt, W. K., Schmidt, G. W. 1996. The effects of elevated temperature on the photosynthetic efficiency of zooxanthellae in hospite from four different species of reef coral, a novel approach. Plant Cell Environ. 19,291 -299.

Warner, M. E., Fitt, W. K., Schmidt, G. W. 1999. Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching. PNAS.96: 8007 - 8012.

Weinbauer, M. G., Velimirov, B. 1995. Biomass and secondary production of the temperate gorgonian coral Eunicella cavolini (Coelenterata: Gorgonacea). Mar. Ecol. Prog. Ser. 121:211–216.

Weinheimer, A.J., Spraggins, R.L. 1969. The occurrence of two new prostaglandin derivatives (15-epi-PGA2 and its acetate, methyl ester) in the gorgonian Plexaura homomalla. Chemistry of coelenterates. Tetrahedron Let. 15: 5185–5188.

Wheaton, J. L., Jaap, W. C. 1988. Corals and other prominent benthic Cnidaria of Looe Key National Marine Sanctuary, Florida. Florida Marine Research Publication No. 43.

Whitehead, L.F., Douglas, A.E. 2003. Metabolite comparisons and the identity of nutrients translocated from symbiotic algae to an animal host. J. Exp. Biol. 206:3149– 3157.

Wilkinson, C. 2002. Status of Coral Reefs of the World. Australian Institute of Marine Science, Townsville.

Wilkinson, C., O. Linden, H. Cesar, G. Hodgson, J. Rubens, and A. E. Stong. 1999. Ecological and socioeconomic impacts of 1998 coral bleaching in the Indian Ocean: an ENSO impact and a warning of future change? Ambio. 28: 188-196.

Wilkinson, C. 1998. The 1997-1998 mass bleaching event around the world. Pages 15-38 in Wilkinson, C. (ed.). Status of Coral Reefs of the World: 1998. Global Coral Reef Monitoring Network.

Williams, E. H. and L. Bunkley-Williams. 1990. The world-wide coral bleaching cycle and related sources of coral mortality. . Res. Bull. 335:1-71.

Williams, D., Andersen, R. J., Van Duyne, G. D., Clardy, J. 1987. Cembrane and pseudopterane diterpenes from the soft coral Gersemia rubiformis. J. Org. Chem. 52: 332-335.

Williams, D. E., Andersen, R. J., Kingston, J. F., Fallis, A. G. 1988. Minor metabolites of the cold water soft coral Gersemia rubiformis. Can. J. Chem. 66: 2928-2934.

160

Wolfe, G., Steinke, M., Kirst, G. 1997. Grazing activated chemical defense in a unicellular marine alga. Nature. 387:894–897.

Wood, R. 2001. Biodiversity and the history of reefs. Geological Journal 36: 251-263.

Wright, A. E., Burres, N. S., Schulte, G. K. 1989. Cytotoxic cembranoids from the gorgonian Pseudopterogorgia bipinnata. Tet. Lett. 30:3491-3494.

Wright, L. L., Cunningham, T. J., Smolen, A. J. 1983. Developmental neuron death in the rat superior cervical sympathetic ganglion:cell counts and ultrastructure. J. Neurocytol. 12:727-738.

Xue, C.-H., Fang, Y., Lin, H., Chen, L., Li, Z.-J., Deng, D., Lu, C.-X., 2001. Chemical characters and antioxidative properties of sulfated polysaccharides from Laminaria japonica. J. App.Phycol. 13:67-70.

Yamamoto, K., Sakata, K., Watanabe, N., Yagi, A., Brinen, L. S., Clardy, J. 1992. Chlorophyllonic acid and methyl ester, a new chlorophyll a related compound isolated as an antioxidant from short-necked clam, ruditapes philippinarum. Tet. Lett. 33:2587- 2588.

Yan, S.J., Chuda, Y., Suzuki, M., Nagata, T., 1999. Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Biosci. Biotech. Biochem. 63:605-607.

Yellowlees, D., Alwyn, T., Rees, V., Leggat, W. 2008. Metabolic interactions between algal symbionts and invertebrate hosts. Plant Cell Envir. 31:679-694.

Yoshioka, P. M., Yoshioka, B.B. 1989. Effects of wave energy, topographic relief and sediment transport on the distribution of shallow-water gorgonians of Puerto Rico. Coral Reefs 8:145-152.

Zhao, H., Joseph, J., Fales, H.M., Sokoloski, E.A., Levine, R.L., Vasquez-Vivar, J., Kalyanaraman, B. 2005. Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proc. Natl. Acad. Sci.102:5727–5732.

Zidorn, C., Schubert, B., Stuppner, H. 2005. Altitudinal differences in the contents of phenolics in flowering heads of three members of the tribe Lactuceae (Asteraceae) occurring as introduced species in New Zealand. Biochem. Syst. Ecol. 33:855–872.

Zidorn, C. and Stuppner, H. 2001. Evaluation of chemosystematic characters in the genus Leontodon. Taxon 50:115–133.

Zu, H., Ming, H., Bannenberg, G.L., Moldéus, P., Shertzer, H.D. 1994. Oxidation pathways for the intracellular probe 2’,7’-dichlorofluorescein. Arch. Toxicol. 68: 71-88.

161 Zu, H., Ming, H., Bannenberg, G.L., Moldéus, P., Shertzer, H.D. 1996. Effects of Glutathioneand pH on the oxidation of biomarkers of oxidative stress. Arch. Toxicol. 70: 628-634.

Zubay, G. L. 1998. Biochemistry: Fourth ed.: Wm C. Brown: Melbourne.

162 APPENDICES

Table A1. GPS coordinates of Pseudopterogorgia sps. collection sites.

Name Latitude Longitude AB102 26 02.641N 77 28.014W Burrows North 26 23.506N 77 46.028W Burrows South 26 20.957N 77 45.034W Cross Harbor Ridge 25 57.341N 77 20.821W Gorda Rock 26 06.963N 77 33.684W Long Rock 26 09.677N 77 34.973W Sandy Point 26 00.170N 77 23.440W Freeport 26 42.250N 79 00.200W Marsh Harbor 26 33.000N 77 03.000W Little San Salvador 24 35.000N 75 58.000W Cat Island 29 09.000N 75 32.000W San Salvador 24 04.000N 74 32.500W Hog Cay 23 37.000N 75 21.000W Bimini 25 44.119N 79 18.460W Pickles 25 00.074N 80 22.400W

Table A2. One-way analysis of variance for FRAP assay of 75% fractions from seven species of the genus Pseudopterogorgia shows that means are significantly different amongst the treatments. (Graphpad Prism)

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 24 F 130.7 R squared 0.9766

ANOVA Table SS df MS Treatment (between columns) 42.22 23 1.836 Residual (within columns) 1.011 72 0.01405 Total 43.23 95

163 Table A3. One-way analysis of variance for FRAP assay of 15 pseudopterosins isolated from Pseudopterogorgia elisabethae shows that means are significantly different amongst the treatments. The water soluble vitamin E analog Trolox serves as the positive control. (Graphpad Prism)

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 15 F 31.24 R squared 0.9358

ANOVA Table SS df MS Treatment (between columns) 1.701 14 0.1215 Residual (within columns) 0.1167 30 0.003889 Total 1.818 44

Table A4. One-way analysis of variance for DCFH-DA - zooxanthellae assay of 75% fractions from seven species of the genus Pseudopterogorgia shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 16 F 7.6 R squared 0.78

ANOVA Table SS df MS Treatment (between columns) 580 17 39 Residual (within columns) 160 32 5.1 Total 750 47

164 Table A5. One-way analysis of variance for DCFH-DA - zooxanthellae assay of 15 pseudopterosins isolated from Pseudopterogorgia elisabethae shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 20 F 52 R squared 0.94

ANOVA Table SS df MS Treatment (between columns) 3900 17 210 Residual (within columns) 240 60 4 Total 4100 79

Table A6. Repeated measures analysis of variance for photoinhibition assay shows that means are significantly different amongst the treatments. Cells with no treatment and cells with solvent only served as controls. (Graphpad Prism)

Repeated Measures ANOVA P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 6 F 80.12 R squared 0.8513

Was the pairing significantly effective? R squared 0.6744 F 69.61 P value < 0.0001 P value summary *** Is there significant matching? (P < 0.05) Yes

ANOVA Table SS df MS Treatment (between columns) 0.3989 5 0.07978 Individual (between rows) 0.9705 14 0.06932 Residual (random) 0.06971 70 0.000996 Total 1.439 89

165 Table A7. One-way analysis of variance for survival of mouse sympathetic neurons deprived of NGF and treated with three concentrations (10, 20 and 40 µM) of pseudopterosin K, ps K 2’ac and ps K 4’ ac shows that means are significantly different amongst the treatments.

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 11 F 73 R squared 0.99

ANOVA Table SS df MS Treatment (between columns) 36000 10 3600 Residual (within columns) 540 11 49 Total 37000 21

Table A8. One-way analysis of variance of ROS quantification using MitoSox Red dye.

One-way analysis of variance P value < 0.0001 P value summary *** Are means signif. different? (P < 0.05) Yes Number of groups 8 F 39 R squared 0.83

ANOVA Table SS df MS Treatment (between columns) 360000 7 51000 Residual (within columns) 74000 56 1300 Total 430000 63

166 Table A9. NMR data for ps K 2’ acetate and 4’ acetate, ps iso- A, C and D. Recorded in CDCl3 at 500 MHz (TMS as internal standard), chemical shifts, multiplicity and coupling constants (J, Hz) were assigned by means of 1H, 13C NMR and 2D NMR data.

Ps K 2’ Ac Ps K 4’ Ac Pos.  H (mult., J in Hz) C  H (mult., J in Hz) C HMBC 1 3.62 (br m) 35.6 3.62 (m) 35.9 11, 13, 14 2α 1.65 (m) 39.6 1.65 (m) 39.8 14 2β 1.67 (m) 1.68 (m) 3 1.55 (m) 30.2 1.57 (m) 30.3 1, 13 4 2.02 (m) 42.7 2.00 (m) 43.1 13 5α 1.12 (m) 28.0 1.11 (m) 28.4 4, 7 5β 2.17 (m) 2.13 (m) 6α 2.10 (m) 30.0 2.11 (m) 30.6 4, 8 6β 1.44 (m) 1.45 (m) 7 3.53 26.9 3.43 27.4 5, 8, 9, 13 (qdd, 7.55, 6.5, 5.3) (qdd, 7.2, 6.6, 5.1) 8 - 133.3 - 133.3 - 9 - 142.1 - 142.6 - 10 - 145.1 - 145.6 - 11 - 121.2 - 121.6 - 12 - 135.7 - 135.4 - 13 - 129.9 - 128.9 - 14 5.09 (d, 9.2) 130.2 5.11 (d, 9.3) 130.1 1, 12, 16, 17 15 - 129.7 - 130.0 - 16 1.67 (s) 25.7 1.68 (s) 26.0 14, 17 17 1.75 (s) 17.8 1.75 (s) 18.0 14, 15, 16 18 1.04 (d, 6.3) 21.1 1.03 (d, 6.0) 21.4 2, 4 19 1.17 (d, 7.1) 24.0 1.17 (d, 6.7) 24.4 6, 8 20 2.02 (s) 10.9 2.02 (s) 11.1 10, 12 1′ 5.07 (d, 4.0) 101.6 5.13 (d, 4.0) 103.2 9, 3′, 5′ 2′ 5.31 (dd, 10.4, 3.6) 72.5 4.04 (dd, 10.3, 4.0) 69.8 3′ 3′ 4.25 (dd, 10.6, 3.6) 69.0 4.35 (dd, 10.4, 3.6) 69.3 2′, 4′ 4′ 3.98 (bd, 3.0) 72.9 5.29 (bd, 3.7) 73.8 3′, C=O 5′ 4.47 (q, 6.5) 67.6 4.48 (q, 7.0) 66.6 1′, 4′, 6′ 6′a 1.36 (d, 6.5) 16.3 1.26 (d, 7.0) 16.6 4′, 5′ OAc 2.28 (s) 21.2 2.19 (s) 21.3 171.3 172.4 7.03 (s) 8.50 (s) 9, 11 OH

167 Pos. Ps Iso-A  Ps Iso-C Ps iso-D H (mult., J in Hz) C H(mult., J in Hz) C H(mult.,Jin Hz) C 1 3.60 (br m) 35.3 3.51 (m) 35.7 3.62 (m) 35.6 2α 1.62 (m) 39.4 1.55 (m) 39.7 1.65 (m) 39.7 2β 1.68 (m) 1.59 (m) 1.66 (m) 3 1.58 (m) 29.8 1.48 (m) 30.1 1.51 (m) 30.0 4 1.99 (m) 43.4 1.96 (m) 43.8 1.99 (m) 43.7 5α 1.06 (m) 28.2 1.05 (m) 28.4 1.05 (m) 28.3 5β 2.14 (m) 2.08 (m) 2.08 (m) 6α 2.12 (m) 30.7 2.08 (m) 31.2 2.08 (m) 31.0 6β 1.48 (m) 1.39 (m) 1.39 (m) 7 3.32 (m) 27.1 3.27 (m) 27.8 3.39 (bq, 6.5) 27.7 8 - 127.0 - 127.9 - 127.9 9 - 144.9 - 145.1 - 145.1 10 - 142.2 - 141.6 - 141.6 11 - 126.9 - 127.3 - 127.3 12 - 134.6 - 135.7 - 135.7 13 - 129.5 - 129.5 - 129.5 14 5.06 (d, 8.9) 130.2 5.02 (d, 9.0) 130.1 5.10 (d, 9.4) 130.1 15 - 128.7 - 129.9 - 129.9 16 1.68 (s) 24.6 1.60 (s) 26.0 1.67 (s) 26.3 17 1.75 (s) 16.5 1.65 (s) 17.9 1.73 (s) 17.9 18 1.04 (d, 6.7) 20.1 0.97 (d, 6.5) 21.4 1.04 (d, 6.1) 21.2 19 1.19 (d, 7.0) 22.3 1.15 (d, 7.0) 23.3 1.23 (d, 7.7) 231 20 2.10 (s) 10.9 2.04 (s) 11.9 2.17 (s) 11.8 1′ 4.36 (d, 7.8) 106.9 4.51 (d, 8.0) 106.2 4.60 (d, 7.8) 105.5 2′ 3.47 (dd, 9.1, 8.0) 74.1 3.77 (dd, 9.0, 8.0) 72.9 3.81 (dd, 9.5, 8.0) 74.5 3′ 3.37 (t, 9.5) 76.6 4.74 (t, 9.5) 79.5 3.75 (t, 9.5) 71.4 4′ 3.56 (dd, 10.0, 5.5) 69.6 3.82 (dd, 10.0, 5.5) 68.7 4.90 (dd, 10.0, 5.5) 81.6 5′a 3.14 (dd, 12.0, 11.0) 66.1 3.21 (dd, 12.0, 66.2 3.25 (dd, 11.0, 63.3 11 0) 10 5) 5′b 3.90 (dd, 11.8, 5.4) 4.00 (dd, 12.0, 5.5) 4.11 (dd, 11.8, 5.5)

OAc - - 2.13 (s) 21.3 2.11 (s) 21.3 - 173.3 171.1 OH - - 7.08 (s) - 6.98 (s) -

168

169

1 Figure A2. H NMR spectrum of pseudopterosin A (17) in CD3OD (500 MHz)

170

1 Figure A3. H NMR spectrum of pseudopterosin B (18) in CDCl3 (500 MHz)

171

1 Figure A4. H NMR spectrum of pseudopterosin C (19) in CDCl3 (500 MHz)

172

1 Figure A5. H NMR spectrum of pseudopterosin D (20) in CDCl3 (500 MHz)

173

1 Figure A6. H NMR spectrum of pseudopterosin G (27) in CDCl3 (500 MHz)

174

1 Figure A7. H NMR spectrum of pseudopterosin I (28) in CDCl3 (500 MHz)

175 1 Figure A8. H NMR spectrum of pseudopterosin J (29) in CDCl3 (500 MHz)

176 1 Figure A9. H NMR spectrum of ps A-D aglycone (26) in CDCl3 (500 MHz)

177

1 Figure A10. H NMR spectrum of ps G-J aglycone (34) in CDCl3 (500 MHz)

178

1 Figure A11. H NMR spectrum of pseudopterosin K (35) in CDCl3 (500 MHz)

179

1 Figure A12. H NMR spectrum of 2’Acetate ps K (36) in CDCl3 (500 MHz)

180

1 Figure A13. H NMR spectrum of 4’Acetate ps K (37) in CDCl3 (500 MHz)

181

1 Figure A14. H NMR spectrum of iso-ps A (30) in CD3OD (500 MHz)

182

1 Figure A15. H NMR spectrum of iso-ps C (32) in CDCl3 (500 MHz)

183

1 Figure A16. H NMR spectrum of iso-ps D (33) in CDCl3 (500 MHz)

184